Chimerical peptidic molecules with antiviral properties against the viruses of the Flaviviridae family

ABSTRACT

The present invention is relative to chimerical peptides, whose primary structure holds at least one segment which inhibits the activation of the NS3 protease of a virus from the Flaviviridae family, they also contain a cell penetrating segment and they are capable of inhibiting or attenuate the viral infection. This invention is also relative to pharmaceutical compounds which contain these chimerical peptides for the prevention and/or treatment of the infection caused by a virus of the Flaviviridae family.

This application is the U.S. National Phase of, and Applicants claimpriority from, International Application Number PCT/CU2007/000020 filed30 Oct. 2007 and Cuban Application bearing Serial No. 2006-0207 filed 30Oct. 2006, which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is relative to the pharmaceutical industry, morespecifically to chimerical peptides, whose primary structure holds atleast a segment which inhibits the NS3 protease of a virus from theFlaviviridae family, they also includes a cell penetrating segment andthey are capable to inhibit or attenuate the viral infection. Theinvention is also relative to pharmaceutical compounds which contain thechimerical peptides for the prevention and/or treatment of the infectioncaused by viruses from the Flaviviridae family.

DESCRIPTIVE MEMORY

The Flaviviridae family is constituted by enveloped positivesingle-stranded RNA viruses, which belong to one of three genera:Flavivirus, Hepacivirus and Pestivirus. The Flavivirus genus includesmore than 70 virus, many of them cause relevant diseases in humans andin other species. Members of this genus are the Yellow Fever Virus(YFV), Dengue Virus (DV), Japanese Encephalitis Virus (JEV), Tick-borneEncephalitis Virus (TBE), West-Nile Virus (WNV), St. Louis EncephalitisVirus (SLEV) and others. The Hepatitis C virus (HCV) is the prototype ofthe Hepacivirus. As members of the genus Pestivirus we find the BovineViral Diarrhea virus (BVDV), the Classical Swine Fever virus (CSFV),Border Disease virus (BDV) and others.

The viruses belonging to different genera of the Flaviviridae family, donot display antigenic cross-reactivity and show diverse biologicalproperties, however they show evident similarities in aspects such asvirion morphology, genome organization and the replication strategy(Leyssen, P., De Clercq, E., Neyts, J. 2000 Perspectives for thetreatment of infections with Flaviviridae. Clin Microbiol Rev., 3:67-82; Rice, C. M. 1996. Flaviviridae: the viruses and theirreplication, p. 931-960. In B. N. Fields, D. M. Knipe, and P. M. Howley(ed.), Fields virology, 3^(rd) ed., vol. 1. Lippincott-Raven Publishers,Philadelphia, Pa.; Westaway, E. G. 1987. Flavivirus replicationstrategy. Adv. Virus Res. 33:45-90)

The HCV constitutes an important health problem worldwide. According tothe WHO about 3% of the world population has been infected by the virus,indicating that more than 170 millions of chronic carriers are in riskof developing cirrhosis and/or liver cancer (Consensus Panel. EASLInternational Consensus Conference on Hepatitis C, Paris, 26-28 Feb.1999, Consensus Statement. J. Hepatol., 1999, 30, 956). Every year about3-4 millions new infections by HCV arise worldwide (Tan, S. L., Pause,A., Shi, Y. & Sonenberg, N. (2002) Nat. Rev. Drug Discov. 1, 867-881).At least 85% of the infected patients evolves to a chronic infection(Alter, M. J., E. E. Mast, L. A. Moyer, and H. S. Margolis. 1998.Hepatitis C. Infect. Dis. Clin. North Am. 12:13-26). The chronicHepatitis C frequently ends in cirrhosis and/or cancer, whether it issymptomatic or asymptomatic. Follow-up studies carried out for 10-20years show development of cirrhosis in 20-30% of the patients, and 1-5%of these patients could develop cancer in the following 10 years (Dutta,U., J. Kench, K. Byth, M. H. Khan, R. Lin, C. Liddle, and G. C. Farrell.1998. Hepatocellular proliferation and development of hepatocellularcarcinoma: a case-control study in chronic hepatitis C. Hum. Pathol.29:1279-1284; Pontisso, P., C. Belluco, R. Bertorelle, L. De Moliner, L.Chieco Bianchi, D. Nitti, M. Lise, and A. Alberti. 1998. Hepatitis Cvirus infection associated with human hepatocellular carcinoma: lack ofcorrelation with p53 abnormalities in Caucasian patients. Cancer83:1489-1494). It is estimated that the number of deaths per year causedby HCV in the United States can reach 35000 by the year 2008 (Dutta, U.,J. Kench, K. Byth, M. H. Khan, R. Lin, C. Liddle, and G. C. Farrell.1998. Hepatocellular proliferation and development of hepatocellularcarcinoma: a case-control study in chronic hepatitis C. Hum. Pathol.29:1279-1284; Pontisso, P., C. Belluco, R. Bertorelle, L. De Moliner, L.Chieco Bianchi, D. Nitti, M. Lise, and A. Alberti. 1998. Hepatitis Cvirus infection associated with human hepatocellular carcinoma: lack ofcorrelation with p53 abnormalities in Caucasian patients. Cancer83:1489-1494.).

Currently, the anti-HCV treatments approved by the FDA are theinterferon monotherapy and the interferon-ribavirin combined therapy(Dymock, B. W. Emerging Drugs 2001, 6(1), 13 and references within).Recently, the use of pegylated interferon variants have been approved,which increase the therapeutic efficacy of these treatments, but theyare still far from being ideals. Because of the seriousness of thisdisease, new and more effective treatments are needed.

The Flavivirus infecting human beings are transmitted by arthropods suchas ticks and mosquitoes, which makes these diseases so difficult toeradicate (Monath, T. P., and F. X. Heinz. 1996. Flaviviruses, p.961-1034. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fieldsvirology, 3rd ed., vol. 1. Lippincott-Raven Publishers, Philadelphia,Pa.). The Yellow Fever is still an important cause of hemorrhagic fever,with mortality rates as high as 50%, although a vaccine is alreadyavailable.

The Dengue Virus is pandemic in tropical areas and its re-emergence isan increasing public health problem worldwide. It is estimated thatannually occur approximately 100 million infections by dengue virus and2.5 milliard persons live in endemic areas (Gubler, D. J. 1998. Clin.Microbiol. Rev. 11, 480-496; Monath, T. P. (1994) Proc. Natl. Acad. Sci.USA 91, 2395-2400.) During the period 1990-1998, the average number ofDengue Hemorrhagic Fever (DHF) cases reported to the WHO was 514 139 peryear, including 15 000 deaths, although it is considered that the realburden of the disease is some times higher. However, neither vaccinesnor specific antiviral treatments are commercially available. The denguevirus complex is conformed by four distinct viruses or serotypes(VD1-VD4), which are related genetically and antigenically. The DV istransmitted to humans by the mosquitoes, mainly by the Aedes aegypti.The infection causes diverse clinical manifestations varying fromasymptomatic and benign to an undifferentiated febrile illness or moresevere manifestations like the DHF and the potentially lethal DengueShock Syndrome (DSS). The most severe clinical manifestations arefrequently associated to sequential infections with two differentserotypes (Halstead, S. B. Neutralization and antibody-dependentenhancement of dengue viruses. Adv. Virus Res. 60:421-67., 421-467,2003. Hammon WMc. New haemorragic fever in children in the Philippinesand Thailand. Trans Assoc Physicians 1960; 73: 140-155). Epidemiologicalstudies have been carried out showing evidences of sequential infectionwith different serotypes as a risk factor for severe disease (Halstead,S. B. Neutralization and antibody-dependent enhancement of dengueviruses. Adv. Virus Res. 60:421-67., 421-467, 2003. Hammon WMc. Newhaemorragic fever in children in the Philippines and Thailand. TransAssoc Physicians 1960; 73: 140-155). This phenomena has been explainedthrough the theory of the “antibody dependent enhancement (ADE)”, whichpostulates that an increase in infectivity is associated with a moreefficient cell entry of the virus mediated by FC receptor of theinfected cells (Halstead S B. Pathogenesis of dengue: challenges tomolecular biology. Science 1988; 239: 476-481).

Other Flavivirus is the JEV, which is the main cause of viralencephalitis worldwide. About 50000 cases occur annually in Asia with ahigh mortality rate of 30% and causing long lasting neurologicaldisorders in 30% of cases (Kalita, J., and U. K. Misra. 1998. EEG inJapanese encephalitis: a clinicoradiological correlation.Electroencephalogr. Clin. Neurophysiol. 106:238-243; Kaluzova, M., E.Eleckova, E. Zuffova, J. Pastorek, S. Kaluz, O. Kozuch, and M. Labuda.1994. Reverted virulence of attenuated tick-borne encephalitis virusmutant is not accompanied with the changes in deduced viral envelopeprotein amino acid sequence. Acta Virol. 38:133-140).

Severe encephalitis is also caused by other Flaviviruses like the TBEV,being two subtypes of this virus: the eastern type with an associatedmortality of 20% and the western type with 1-2% (Heinz, F. X., and C. W.Mandl. 1993. The molecular biology of tick-borne encephalitis virus.APMIS 101:735-745); the Murray Valley Encephalitis (MVE) in Australia(Mackenzie, J. S., and A. K. Broom. 1995. Australian X disease, MurrayValley encephalitis and the French connection. Vet. Microbiol.46:79-90); the SLEV in the western United States and the WNV, which isendemic in Africa, Middle East and the Mediterranean and has also causedrecent outbreaks in the United States. Since it appeared in the UnitedStates in 1999, it has expanded very fast, infecting about 15 000persons and leading to more than 600 deaths. However, currently thereare not available vaccines or drugs which protect against the WNV (vander Meulen, K. M., Pensaert, M. B. and Nauwynck, H. J. (2005) West Nilevirus in the vertebrate world. Arch. Virol. 150, 637-657).

Hemorrhagic manifestations are caused by other Flaviviruses like theOmsk Hemorrhagic Fever Virus (OHFV) in Russia, with a lethality ratebetween 0.5-3% and the Kyasanur Forest Disease Virus (KFDV) in India(Monath, T. P., and F. X. Heinz. 1996. Flaviviruses, p. 961-1034. In B.N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rded., vol. 1. Lippincott-Raven Publishers, Philadelphia, Pa.).

Other Flavivirus, the Louping ill virus (LIV) infects mainly sheep,although occasional human infections have also been reported (Davidson,M. M., H. Williams, and J. A. Macleod. 1991. Louping ill in man: aforgotten disease. J. Infect. 23:241-249).

The Pestivirus BVDV, CSFV and BDV cause important diseases in animals.In their respective hosts the cause severe affections which usually leadto death, although these viruses can cross species causing a milderdisease in other hosts. Frequently, infections occur by oronasal ortransplacental route. The latter is responsible of persistent infectionswhich are a threat for the rest of the livestock (Edwards, S., P. M.Roehe, and G. Ibata. 1995. Comparative studies of border disease andclosely related virus infections in experimental pigs and sheep. Br.Vet. J. 151:181-187).

It is presumed that the members of the Flaviviridae family share asimilar replication strategy. The viral replication cycle begins withthe adhesion of the virus to the host cell surface. In the case ofDengue virus, it has been shown that the virus binds toglycosaminoglycans, which could be the initial site of interaction withthe cells. It has been also shown that the virus binds to DC-SIGN,although it is likely that the role of these molecules is related to theviral concentration on the cell surface or in the spread of the virus tosecondary replication sites in vivo. After the initial binding, thevirus interacts with high affinity receptors and/or co-receptors, whichmediate the virus entry into the cell by endocytosis. In the case of theWNV, it has been postulated that the α_(v)β₃ integrin could serve tothese means (Chu, J. J-H., and Ng, M.-L., 2004. Interaction of West NileVirus with α _(vβ) ₃ Integrin Mediates Virus Entry into Cells. J. Biol.Chem. 279, 54533-54541). It has also been shown that the HCV binds tothe cellular receptor CD81 (Pileri, P., Y. Uematsu, S. Campagnoli, G.Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G.Grandi, and S. Abrignani. 1998. Binding of hepatitis C virus to CD81.Science 282:938-941). Once the virus is localized in the endocyticcompartments, a drop in the compartment pH induces the fusion processbetween the viral and the cell membrane, and this process is mediated bystructural changes of the fusion protein of the virus envelope. Thisprocess leads to the discharge of the virus capsid into the cytoplasm,where the viral RNA is later released.

In the cytoplasm the genomic RNA of the virus, interacts through itsnon-coding 5′ region (5′UTR) with the ribosome, leading to thetranslation of the virus unique open reading frame. This way, theprecursor viral polyprotein is synthesized, which in the case ofFlaviviruses includes three structural proteins (C, preM and E) and fivenon structural proteins (NS1-5). This polyprotein is then modified co-and post-translationally giving rise to the individual mature functionalproteins of the virus. The RNA-dependent RNA-polymerase of the viruswith associated cofactors produces copies of negative-single strandedRNA, which are later used as templates for the synthesis of the genomicpositive-single stranded viral RNA. The viral proteins participating inthe replication are associated to membranous structures apparentlyrelated to the endoplasmic reticulum (ER).

After the replication is completed, the genomic RNA associates with thenucleocapsid, the immature virions bud into the lumen of the ER (buddingoccurs at the membrane of the ER or related membranous structuresinduced by the virus), covered by a lipid envelope containing viralproteins. Passing through the exocytic pathway, the envelope proteinsare glycosilated and become mature, leading to the final release of themature virions to the extracellular space.

The replication of the Flaviviriae requires the NS3pro protease(localized approximately in the first 180 residues of the non structuralprotein NS3) for the correct processing of the precursor polyprotein,this way constituting an attractive potential target for the design ofantiviral drugs (Chappell, K. J., Nall, T. A., Stoermer, M. J., Fang, N.X., Tyndall, J. D., Fairlie, D. P. and Young, P. R. (2005) Site-directedmutagenesis and kinetic studies of the West Nile Virus NS3 proteaseidentify key enzyme-substrate interactions. J. Biol. Chem. 280,2896-2903. SHIRYAEV, S. A., RATNIKOV, B. I., CHEKANOV, A. V., SIKORA,S., ROZANOV, D. V., GODZIK, A., WANG, J., SMITH, J. W., HUANG, Z.,LINDBERG, I., SAMUEL, M. A., DIAMOND, M. S. and Alex Y. STRONGIN, A. Y.,2006. Cleavage targets and the D-arginine-based inhibitors of the WestNile virus NS3 processing proteinase. Biochem. J. 393, 503-511.Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M. J. Virol.2000, 74, 2046; Bartenschlager, R.; Lohmann, V. J. Gen. Virol. 2000, 81,1631. Matusan, A. E., Kelley, P. G., Pryor, M. J., Whisstock, J. C.,Davidson, A. D. and Wright, P. J. (2001) J. Gen. Virol. 82, 1647-1656).

In Flavivirus this protease is responsible for the proteolytic cleavageat the junctions NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4N/NS5, as well asthe internal cleavage in C, NS3 and NS4A (Chambers, T. J., Nestorowicz,A., Amberg, S. M. and Rice, C. M. (1993) Mutagenesis of the yellow fevervirus NS2B protein: effects on proteolytic processing, NS2B-NS3 complexformation, and viral replication. J. Virol. 67, 6797-6807. Jan, L. R.,Yang, C. S., Trent, D. W., Falgout, B. and Lai, C. J. (1995) Processingof Japanese encephalitis virus non-structural proteins: NS2B-NS3 complexand heterologous proteases. J. Gen. Virol. 76, 573-580. Lobigs, M.(1993) Flavivirus premembrane protein cleavage and spike heterodimersecretion require the function of the viral proteinase NS3. Proc. Natl.Acad. Sci. U.S.A. 90, 6218-6222. Yamshchikov, V. F. and Compans, R. W.(1994) Processing of the intracellular form of the west Nile viruscapsid protein by the viral NS2B-NS3 protease: an in vitro study. J.Virol. 68, 5765-5771).

In HCV, NS3pro mediates the proteolytic processing of the viralpolyprotein in the segment comprised between the proteins NS2-NS5B (R.Bartenschlager, 1999, The NS3/4A proteinase of the hepatitis C virus:unravelling structure and function of an unusual enzyme and a primetarget for antiviral therapy. J. Viral Hepat. 6, 165).

Besides the central role played by the NS3pro protease in the viralreplication cycle processing the virus proteins, this protein can alsoprocess cellular substrates and hence it could be involved in variousmechanisms of cellular damage and pathogenesis (Shiryaev, S. A.,Ratnikov, B. I., Chekanov, A. V., Sikora, S., Rozanov, D. V., Godzik,A., Wang, J., Smith, J. W, Huang, Z., Lindberg, I., Samuel, M. A.,Diamond, M. S, and Strongin, A. Y. (2005) The cleavage targets and the(D)-arginine-based inhibitors of the West Nile virus NS3 processingproteinase. Biochem. J. 393, 503-511).

Thus, it has been shown that the NS3 protease from WNV producesproteolytic cleavage in neuronal myelin basic protein (MBP). RegardingDV and WNV, it has been suggested that NS3 is involved in the inductionof virus mediated apoptosis (Ramanathan, M. P., Chambers, J. A.,Pankhong, P., Chattergoon, M., Attatippaholkun, W., Dang, K., Shah, N.and Weiner, D. B. (2005) Virology doi:10/1016/j.virol. 2005.08.043)

For its optimal function, the NS3 protease needs to interact with otherviral protein or cofactor, the NS2B protein in Flavivirus and NS4A inHepacivirus and Pestivirus. In DV the presence of NS2B induces anincrease in the proteolytic activity of NS3 between 3300 and 6600 times(Yusof, R., Clum, S., Wetzel, M., Murthy, H. M. & Padmanabhan, R., 2000.J. Biol. Chem. 275, 9963-9969).

In HCV, NS3 binding to NS4A is required for the proteolytic cleavage atNS3/4A, NS4A/B and NS4B/5A and it increases the efficiency of theprocessing at the junction NS5A/B (Bartenschlager R, Ahlborn L L, MousJ, Jacobsen H. Kinetic and structural analyses of hepatitis C viruspolyprotein processing. J Virol 1994; 6: 5045-5055. Fulla C, Tomei L, DeFrancesco R. Both NS3 and NS4A are required for proteolytic processingof hepatitis c virus nonstructural proteins. J Virol 1994; 6: 3753-3760.Lin C, Pragai B M, Grakoui A, Xu J, Rice C M. Hepatitis C virus NS3serine proteinase: trans-cleavage requirements and processing kinetics.J Virol 1994; 6: 8147-8157. Tanji Y, Hijikata M, Satoh S, Kaneko T,Shimotohno K. Hepatitis C virus-encoded nonstructural protein NS4A hasversatile functions in viral protein processing. J Virol 1995; 6:1575-1581). The addition of a NS4A fragment to NS3pro in a 10 timesmolar excess increases the catalytic efficiency coefficientK_(cat)/K_(m) in approximately 40 times (SHIMIZU, Y., YAMAJI, K.,MASUHO, Y., YOKOTA, T., INOUE, H., SUDO, K., SATOH, S. y SHIMOTOHNO, K.1996. Identification of the Sequence on NS4A Required for EnhancedCleavage of the NS5A/5B Site by Hepatitis C Virus NS3 Protease. J. Virol70, 127-132).

The crystal structures of NS3pro and the NS3pro-NS2B complex from DV andthe complex formed by NS3pro-NS2B from WNV with a peptide inhibitor havebeen experimentally determined (Murthy, H. M., Clum, S. & Padmanabhan,R., 1999. J. Biol. Chem. 274, 5573-5580. Murthy, H. M., Judge, K.,DeLucas, L. & Padmanabhan, R., 2000. J. Mol. Biol. 301, 759-767. ErbelP, Schiering N, D'Arcy A, Renatus M, Kroemer M, Lim S P, Yin Z, Keller TH, Vasudevan S G, Hommel U., 2006. Structural basis for the activationof flaviviral NS3 proteases from dengue and West Nile virus. Nat. StructMol. Biol.). Similarly, the crystal structures of NS3pro and the complexNS3pro/NS4A from HCV have also been determined (Love, R. A., Parge, H.E., Wickersham, J. A., Hostomsky, Z., Habuka, N., Moomaw, E. W., Adachi,T., Hostomska, Z., 1996. The crystal structure of hepatitis C virus NS3proteinase reveals a trypsin-like fold and a structural zinc bindingsite. Cell. 87, 331-342. Kim, J. L., Morgenstern, K. A., Lin, C., Fox,T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre,C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A.,Caron, P. R., Thomson, J. A., 1996. Crystal structure of the hepatitis Cvirus NS3 protease domain complexed with a synthetic NS4A cofactorpeptide. Cell. 87, 343-535. Erratum in: Cell, 89:159, 1997).

The NS3pro protease adopts a chymotrypsin-like fold, which comprises twobeta barrels and the His51-Asp75-Ser135 catalytic triad being localizedin a crevice created between these domains. The binding of NS2B proteininduces large changes in the tridimensional structure of NS3pro,affecting both the N- and C-terminal domains and comprising changes inthe location and extend of the secondary structural segments.

The structure of the complex formed by the NS3pro-NS2B active proteasewith a peptide inhibitor shows that NS2B forms a belt around NS3pro,adopting a mainly extended structure and including five beta strands.

The first three strand are associated to beta strands from NS3 protein:the strand Trp53-Ala58 (WNV numbering) runs antiparallelly to the NS3beta strand Gly21-Met26 corresponding to the N-terminal beta barrel andthe beta strands Glu67-Ile68 and Arg74-Asp76 are parallel to the betastrands B2a y B2b of the NS3 C-terminal beta barrel. The strands 4 and 5form a beta hairpin, which interacts with the substrate binding sitecontacting the E1b-F1 loop from the N-terminal beta barrel. The foldingof NS2B below the E2b-F2 beta hairpin of the C-terminal barrel induces aconformational change in this region of NS3 which leads to thearrangement of residues important for substrate recognition (Gly151,Gly153 y Tyr161). The residue Tyr161 makes pi-cation interactions withthe arginine at P1 position. The negative electrostatic potentialassociated to the main chain carbonyl groups of residues Asp82-Gly83 andthe atom Od1 of Asn84 from NS2B makes it favorable the interaction withthe positive charge of the guanidinium group from the arginine atposition P2. This way, they contribute to the conformation of the S2site. Thus, the NS2B binding to NS3 completes essential elements of theenzyme active site and it also contributes to the thermodynamicstability of protein folding. These facts offer a structural basis forunderstanding the activation process of this protease.

In the case of HCV, NS3 activation is mediated by the binding of thebeta strand Thr20-Leu31 from NS4A, which is structurally equivalent tostrand 1 of NS2B from Flavivirus (SHIMIZU, Y., YAMAJI, K., MASUHO, Y.,YOKOTA, T., INOUE, H., SUDO, K., SATOH, S. y SHIMOTOHNO, K. 1996.Identification of the Sequence on NS4A Required for Enhanced Cleavage ofthe NS5A/58 Site by Hepatitis C Virus NS3 Protease. J. Virol 70,127-132).

Among the current approaches being carried out to obtain antiviralmolecules against Flaviviridae, those based on NS3 inhibition arefocused mainly in developing inhibitors targeting the active site. Theseapproaches seem to be very promising, which is supported by recentresults achieved in the development of drugs against HCV. However, theseexperiences have also shown clearly the difficulties inherent to theseapproaches. One of the more prominent is the generation of escapemutants. The polymerases of RNA viruses have relatively low fidelity andin the case of HCV it introduces a mutant per copy of the virus genome.It results in the fact that molecules developed by this means althoughthey are very potent they could have a limited useful lifetime. It haslead to the introduction of therapeutic interventions based on cocktailsof drugs as a need for antiviral treatments. It has also been observedthat escape mutants raised against one drug can frequently escape theantiviral activity of other drugs targeting the same active site.

The present invention describes novel methods aimed to the design ofantiviral agents against Flaviviridae, which are based in the concept ofinhibition of the NS3 protease activation process. The key approach ofthis concept is the design of peptidic molecules and/or drugs capable ofblocking the interaction between NS3 and its cofactor (NS2B or NS4A),and hence being able to interfere with the correct folding of the activeNS3 protease. Such molecules are capable to bind to regions of the NS3protease which are involved in the interaction with the cofactor, andcompete with it and/or stabilize the structure of the inactive protease.

An advantage of this invention is that the probability of generation ofescape mutants against these molecules is expected to be lower comparedto those inhibitors of the active protease which compete with thesubstrate for the active site. The molecules of the present inventionbind to binding sites in NS3 which are involved in protein-proteininteractions essential for the viral replication cycle, thereforemutations generated in these regions of NS3 should have additionalcompensatory mutations in the cofactor.

Other advantage is the high specificity of the inhibitory activitydisplayed by these molecules. It is due to the fact that its bindingsites on NS3 are essentially specific for the viral protease and theyare not present on the host serine proteases. Furthermore, the hostserine proteases have active sites showing specificities very similar toNS3 and hence they could be potential targets for toxicity of activesite blocking drugs.

In the present invention we describe chimerical peptidic molecules whichinhibit infections by Flaviviridae, and whose primary structure can bedescribed according the following formula:[P]-[L₁]-[I]-[L₂]-[T] or [I]-[L₃]-[P]-[L₄]-[T],where, [P] is the amino acid sequence of a “cell penetrating peptide”,typically of 10-30 amino acids, which have the capacity to allow theinternalization of the whole peptidic molecule into the cell cytoplasmand to get access to the contiguity of the rough endoplasmic reticulum(RER); [L1, L2, L3, L4], are linker sequences of 0-6 residues; [I], is aNS3pro activation inhibitor sequence, containing residues which makecontacts with at least one amino acid from the beta strands B2a and B2bof the C-terminal beta barrel, or from the beta strand A1 of theN-terminal beta barrel of the NS3pro protein from Flavivirus (or thecorresponding structurally equivalent regions of Pestivirus orHepacivirus) in its active or inactive conformation; [T], amino acidsequence between 0 and 10 residues, which is typically one or twosignals of retention in the ER (like the sequences KDEL, KKXX andLRRRRL), or the sequence XRR with the capability to bind the P1 and P2substrate binding sites of the NS3pro protease of Flavivirus.

More specifically, we have shown that peptides which have been designedaccording to the present invention are capable of inhibit the viralinfection by DV.

Cationic Cell Penetrating Peptides

The present invention describes the design of chimerical peptides whichare capable to inhibit the viral infection of viruses from theFlaviviridae family. The designed peptides contain an [I] segment, whichinhibit the activation of the viral NS3pro protease. However, in thisinvention we show that synthetic peptides with amino acid sequencescorresponding to the segment [I] are not capable to penetrate the targetcells and hence they do not inhibit the viral infection in cell linesand in vivo. Inhibition of the viral infection is achieved combining the[I] segment with a cell penetrating [P] segment.

A number of peptides derived from certain proteins have the capabilityto penetrate into the cells and get access into the cytoplasm andnucleus. These peptides are known as cell penetrating peptides orprotein transduction domains (PTD) (Joliot, A., and Prochiantz, A.(2004) Transduction peptides: from technology to physiology. Nat. CellBiol. 6, 189-96. Snyder, E. L., and Dowdy, S. F. (2004) Cell penetratingpeptides in drug delivery. Pharm. Res. 21, 389-93. Deshayes, S., Morris,M. C., Divita, G., and Heitz, F. (2005) Cell penetrating peptides: toolsfor intracellular delivery of therapeutics. Cell. Mol. Life. Sci. 62,1839-49). The most studied PTDs are the cationic peptides derived fromproteins such as the HIV transcription factor TAT, the homeoboxantennapedia (penetratin) from Drosophila melanogaster and the proteinVP22 from the Herpes simplex virus. These peptides have raised greatinterest as potential carriers for the introduction of cargo moleculesinto the cells in order to enhance their biological activity, beingthese cargoes very diverse in nature like small drug-like molecules orgenes and proteins. The potential of the PTDs as vectors for moleculeswith therapeutic interest have been shown in cell systems and also inanimal models (Beerens, A. M., Al Hadithy, A. F., Rots, M. G., andHaisma, H. J. (2003) Protein transduction domains and their utility ingene therapy. Curr. Gene Ther. 3, 486-94. Wadia, J. S., and Dowdy, S. F.(2003) Modulation of cellular function by TAT mediated transduction offull length proteins. Curr. Protein Pept. Sci. 4, 97-104.) Wadia, J. S.,and Dowdy, S. F. (2005) Transmembrane delivery of protein and peptidedrugs by TAT-mediated transduction in the treatment of cancer. AdV. DrugDeli Very ReV. 57, 579-96. Rudolph, C., Schillinger, U., Ortiz, A.,Tabatt, K., Plank, C., Muller, R. H., and Rosenecker, J. (2004)Application of novel solid lipid nanoparticle (SLN)-gene vectorformulations based on a dimeric HIV-1 TAT-peptide in vitro and in vivo.Pharm. Res. 21, 1662-9).

A significant amount of research have been carried out in order toelucidate the mechanisms by which these peptides can get access into thecytoplasm and the nucleus passing through biological barriers formed bythe cellular membrane systems such as the plasma membrane, the membranesof the endocytic compartments and the nucleus. Recently, it has beenshown that a number of previously documented observations in culturecells regarding the cellular localization and cell entry of the PTDs atlow and physiological temperature were due to artifacts caused by thefixation procedues and unspecific binding of peptides to the plasmamembrane (Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure,B., Gait, M. J., Chemomordik, L. V., and Lebleu, B. (2003) Cellpenetrating peptides. A reevaluation of the mechanism of cellularuptake. J. Biol. Chem. 278, 585-90. Vives, E., Richard, J. P., Rispal,C., and Lebleu, B. (2003) TAT peptide internalization: seeking themechanism of entry. Curr. Protein Pept. Sci. 4, 125-32).

The most recent results suggest that endocytosis plays an essential rolein the entry of PTDs into the cells. However, a detailed and generallyaccepted description of the intracellular traffic of these peptides hasnot emerged yet.

It was first reported that TAT peptide fusion proteins entry into thecells passing to neutral caveosomes via plasma membrane caveolae, butmore recent studies have shown that caveolae are not required and TATpeptide cell entry occurs by macropinocytosis (Ferrari, A., Pellegrini,V., Arcangeli, C., Fittipaldi, A., Giacca, M., and Beltram, F. (2003)Caveolae-mediated internalization of extracellular HIV-1 tat fusionproteins visualized in real time. Mol. Ther. 8, 284-94. Wadia, J. S.,Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA fusogenicpeptide enhances escape of TAT-fusion proteins after lipid raftmacropinocytosis. Nat. Med. 10, 310-5). Consistently with the postulatedcell entry mediated by endocytosis, the PTDs have been observed in earlyand recycling endosomes. However, the biological activity shown by themolecules associated to the PTDs indicates that these peptides shouldescape at least partially from the endocytic compartments by a stillunknown mechanism getting access into the cytosol. Colocalization ofinternalized TAT peptide with the Golgi marker BODIPY-ceramide has beenreported, consistently with its lacks of visualization in laterendosomes and lysosomes labeled with Lysotracker (Fischer, R., Kohler,K., Fotin-Mleczek, M., and Brock, R. 2004. A stepwise dissection of theintracellular fate of cationic cellpenetrating peptides. J. Biol. Chem.279, 12625-35).

These data suggest that these peptides are capable to traffic to theGolgi directly from the early endosomes, which is consistent with apotential peptide entry into the cytosol from the ER preceded byretrograde transport of the peptides from the Golgi. However, otherstudies have reported colocalization of peptides in acidic lateendocytic structures and in lysosomes. Such results have been reportedfor TAT peptide, octaarginine, TAT protein and conjugates of liposomeswith TAT peptide (Al-Taei, S., Penning, N. A., Simpson, J. C., Futaki,S., Takeuchi, T., Nakase, I., and Jones, A. T. 2006. IntracellularTraffic and Fate of Protein Transduction Domains HIV-1 TAT Peptide andOctaarginine. Implications for Their Utilization as Drug DeliveryVectors. Bioconjugate Chem. 17, 90-100. Fretz, M. M., Koning, G. A.,Mastrobattista, E., Jiskoot, W., and Storm, G. (2004) OVCAR-3 cellsinternalize TAT-peptide modified liposomes by endocytosis. Biochim.Biophys. Acta 1665, 48-56. Vendeville, A., Rayne, F., Bonhoure, A.,Bettache, N., Montcourrier, P., and Beaumelle, B. (2004) HIV-1 Tatenters T cells using coated pits before translocating from acidifiedendosomes and eliciting biological responses. Mol. Biol. Cell 15,2347-60).

However, it is possible that the PTDs could exploit various differentmechanisms of cell entry and intracellular traffic, depending of severalfactors like cell type, nature of the PTD, temperature, cargo, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes two topological variants of chimericalpeptides which inhibit the viral infection by Flaviviridae:[P]-[L₁]-[I]-[L₂]-[T] o [I]-[L₃]-[P]-[L₄]-[T],

As [P] penetrating peptide are preferably selected, but not restrictedto those, cationic peptides with the capability to carrier cargomolecules into the cells. As possible cationic peptides could be chosenpenetratin, polyarginines of 7-10 residues such as R9 nonapeptide or R10decapeptide or TAT peptide, although any other peptide sequence between10-30 residues showing similar penetrating capability could be selected.These penetrating cationic peptides have the capability to penetrateinto the cytoplasm of the cell via endocytosis, which could involve thetraffic through the ER. This property is favorable for the biologicalactivity of these peptides because it guarantees the peptidelocalization in the contiguity of the RER, the place where the precursorpolyprotein synthesis and processing is carried out and constitutes thetarget of peptide antiviral activity.

Alternatively, other cell penetrating peptides could be also used as [P]segments like the cationic dendrimeric peptides or peptides comprisingD-amino acids, which are very resistant to proteolytic degradation. Thecationic peptides also guarantee a good biodistribution in vivo of thepeptides from the present invention, allowing its favorable effectiveconcentration in organs and tissues infected by Flaviviridae, to higherlevels compared to larger molecules as the monoclonal antibodies. Oneexample could be the use of peptides permeable to the blood-brainbarrier (BBB) to treat Flaviviridae infections causing encephalitis likeTBE, WNV, JEV, SLEV and KV. The molecular transport through the BBB is aformidable problem even for small drugs aimed for treatment ofintraencephalic diseases (Temsamani, J. and Vidal, P. 2004. The use ofCell-penetrating peptides for drug delivery. Drug Discov. Today 9,1012-1019).

The NS3pro protease inhibitory sequence [I], has the capability toinhibit or modify the interaction between the proteins NS3 and NS2B fromFlavivirus (or between NS3 and NS4A from Hepacivirus and Pestivirus),and this way it affects the correct folding of NS3pro which is necessaryfor the protease activation process. In one embodiment of thisinvention, [I] consists in the sequence Asp50-Glu62 of the protein NS2Bfrom DV2, or its homologous sequences from other Flavivirus. Thissequence contains the residues corresponding to the beta strand 1 of theprotein NS2B, which makes contacts with residues located at theN-terminal beta barrel of the active NS3pro protein. Thus, peptidesaccording to the topologies described in this invention, compete withthe native sequence of the cofactor NS2B protein during the folding ofthe NS3pro protein to the adoption of its active conformation. It leadsto the formation of inactive NS3pro-peptide complexes because the fullactivation requires structural rearrangements not only in the N-terminaldomain, but also in the C-terminal beta barrel two. Protease activationwould need an additional binding of the region Glu66-Ile86 of theprotein NS2B to the C-terminal domain of NS3pro. In addition, thebinding of the segment [I] serves as an anchor of the peptides of thepresent invention to the protein NS3, in such a way that the N- orC-terminal extensions of these peptides could alter the surfacetopography of NS3 and interfere with its interactions with viral and/orhost proteins. Such interactions include the substrate recognitionand/or other interactions related to the conformation and/or functioningof the viral replication complex. Thus, in one embodiment of the presentinvention the [P] segment corresponding to the first topological variantare poly-D arginine, which besides having the cell penetrating propertyare also inhibitors of the Flavivirus NS3pro protease (SHIRYAEV, S. A.,RATNIKOV, B. I., CHEKANOV, A. V., SIKORA, S., ROZANOV, D. V., GODZIK,A., WANG, J., SMITH, J. W., HUANG, Z., LINDBERG, I., SAMUEL, M. A.,DIAMOND, M. S. and Alex Y. STRONGIN, A. Y., 2006. Cleavage targets andthe D-arginine-based inhibitors of the West Nile virus NS3 processingproteinase. Biochem. J. 393, 503-511). Thus, the binding of the [I]segment/anchor to NS3pro facilitates the fit of the polyArg peptides inthe substrate binding site of the protease, well corresponding to thesame chain anchored by the peptide (cis-inhibition) or a different chain(trans-inhibition). In an analog way, Hepacivirus and Pestivirusinhibitory peptides incorporate as [I] sequence the segmentcorresponding to the region Thr20-Leu31 of the NS4A protein (numberingof HCV), which is structurally equivalent to the beta strand 1 of theNS2B protein from Flavivirus.

In a second embodiment, which applies to Flavivirus, the [I] segmentdoes not relate to any specific segment of the NS2B sequence, butconsists in a peptide sequence with the capability to bind to NS3proprotein and stabilize the N-terminal barrel in its inactiveconformation. In this case the peptide sequence makes contacts with thesegment corresponding to Tyr23-Tyr33 of the NS3pro protein from DV2, ora homologous region corresponding to other Flavivirus. In addition, the[I] segment also makes stabilizing structural contacts with the residuesfrom the segments Ala1-Gly14 and Ala56-Met59 of the protein NS3pro.Therefore, these peptides promote their inhibitory effect by interferingwith the native folding of the protein NS3, inducing a folding pathwayleading to the inactive conformation of the protease.

Such [I] sequences could be obtained by theoretical methods and/orexperimental methods which make use of combinatorial libraries. In caseof design by theoretical methods, the invention implies the use of oneor various methods of computational molecular modeling and the use ofthree dimensional structural models of the protein NS3pro in itsinactive conformation. Making use of the method(s) of computationalmodeling and the spatial coordinates of the 3D structural model of theinactive NS3pro protein, it is possible to model a polypeptide mainchain in an extended conformation, which forms an antiparallel betastrand with the segment corresponding to the beta strand A1 of theN-terminal beta barrel. In addition, it is also possible to model theside chains of the polypeptide chain, in such a way that the chemicalidentity of this side chains and its conformers imply energeticallyfavorable atomic contacts. This invention involves the combinedexploring by computational means of the peptide sequence andconformational space, the side chain rotamer space of the peptide andalso of the protease and the selection of the most favorable peptidevariants according to an energy scoring of the obtained models, whichindicate a potential higher affinity of the peptide-protein interaction.

The coordinates corresponding to the inactive NS3pro structural modelscould originate from experimental data obtained by the methods of x-raydiffraction and/or NMR or by the use of models obtained by computationalmodeling methods. In the case of DV2, the coordinates could be obtainedfrom the file 1BEF of the Protein Data Bank (PDB). For other Flavivirus,it is possible to obtain 3D models by the method of homology modeling.In the present invention we describe the [I] sequence: QWPALPKIEAQDG(SEQ ID NO: 369), which was designed according to this second embodimentof the present invention. The FIG. 1D shows a computational model of thetridimensional structure of the NS3pro-[I] complex corresponding to thisembodiment. According to this model, the [I] segment adopt an extendedbeta strand structure associated to the segment Gly29-Y33 of NS3pro (DV2numbering).

Additionally, combinatorial libraries of synthetic peptides or phagedisplayed peptides libraries could be used in order to obtain [I]sequences with similar properties to peptides described in the secondembodiment of the present invention. In this case the recombinant NS3proprotein is used as target for ligand selection or biopanning.

In other embodiment of the present invention, the [I] segment consist inthe sequence Ser70-Gly82 of the protein NS2B from DV2, or its homologoussequences from other Flavivirus. This sequence contains the beta strands3 and 4 of the protein NS2B, which contact and form part of the activeNS3pro protease. Thus, peptides according to the topologies described inthis invention, compete with the corresponding segment of the cofactorNS2B protein during the folding of the NS3 protein to its activeconformation and hamper the proteolytic processing at the junctionNS2B-NS3. It leads to the formation of inactive NS3pro-peptidecomplexes, because these peptides interfere with the correctconfiguration of the substrate binding site, in particular at P2 site,which is essential for the enzyme catalytic activity.

In addition, the binding of the [I] segment corresponding to peptides ofthis invention serves as an anchor to the protein NS3, such that theN-terminal or C-terminal extension of the peptides could modify thesurface topography of NS3 and interfere with the interaction of thisprotein with other viral or host proteins.

In an embodiment related to the previous one (peptide 10 in table 1),the [I] segment consists in the sequence Ser70-Ile86 of the NS2B proteinfrom DV2, or its homologous sequences from other Flavivirus. This regionincludes, besides the beta strands 3 and 4, also the beta strand 5 ofNS2B. In this case, the peptides corresponding to the first topologicalvariant include a C-terminal extension comprising a [L2] segment of 3 or4 residues and a [T] segment consisting of the tripeptide XRR, with aC-terminal carboxylic group. The sequences of these peptides areconsistent with their binding to the NS3 protein adopting its activeconformation, the beta strand 5 and the loop between the strands 4 and 5guarantee the correct formation of the P2 site.

Moreover, the binding of the segment [I] facilitates the structuralchanges in the C-terminal beta barrel which are necessary for theactivation, such as the change in orientation of the E2b-F2 betahairpin, which allows the arrangement of important residues involved inthe substrate recognition like Gly151, Gly153 and Tyr161. However, theformed complex is inactive, because the [L2] segment serves asstabilizing-linker allowing the additional binding of the [T] segment tothe substrate binding site, with the dipeptide RR occupying thepositions S1 and S2. Thus, the protease active site becomes blocked bythe peptide. The segments [L1], [L2], [L3] and [L4] of the presentinvention are linker sequences of 0-6 residues, which connect thesegment [P], [I] and [T], depending on the topological variant. Theselinker segments contains mainly small and/or polar amino acids (Gly,Ser, beta-Ala), which provide flexibility. These linker segments couldalso consist of sequences capable to interact favorably with residuesfrom the NS3pro protein, providing the peptides of the present inventionwith an additional stabilizing effect.

The [T] segments of the present invention are sequences between 0 and 10amino acids, localized at the C-terminal ends of the peptides. In anembodiment, the [T] segment is an ER retention signal, like the KDELsequence. The addition of this signal facilitates the traffic ofpeptides by retrograde transport to the ER. The increase in peptideconcentration within the ER contributes to enhance the transport of thepeptides to the cytosol. It results in an increase of the effectivepeptide concentration in the contiguity of the ER, where occurs thesynthesis of the viral polyprotein and in particular the synthesis ofthe NS3pro. The incorporation of the KDEL signal to the peptide sequenceis compatible with the presence of cationic cell penetrating peptides as[P] segments, because the retrograde transport through the ER is aputative pathway of cationic peptide penetration into the cytosol. Thisway of penetration involved the traffic of peptides from early endosomesto the ER via the trans-Golgi network (TGN). The sequence KDEL interactswith the KDEL receptor present at the TGN which transport the peptide tothe ER where it is discharged. The peptide transport from the ER lumeninto the cytosol is an efficient process, which occurs through channelspresent at the ER membrane formed by the Sec61 protein from thetranslocon complex. This mode of penetration into the cytosol isexploited by bacterial toxins like the cholera toxin, Ricin and theexotoxin A from Pseudomonas, etc.

Use of a FG Hairpin Based Segment as Cell Penetrating Peptide.Inhibitory Effect of This Segment on DV Entry into the Cell.

A novelty of the present invention is the modular structures displayedby these peptides, combining segments or modules with differentfunctions: segment with antiviral activity, cell penetrating peptide,signals for traffic and intracellular localization, lipidation, etc.Thus, it is possible to exploit the capability of peptides of 20-30residues to incorporate in its sequence a great variety of information,which allows to maximize the functional activity of the peptides incells and in vivo. In this invention we have also incorporated bi- orpoly-functional modules in peptide design.

The antiviral activity displayed by peptides of the present invention isbased primarily on the inhibition of NS3 protease activation process.The [I] segments or modules described in the present invention asinhibitors of the viral protease activation process, have the capabilityto bind to the NS3pro protein and to block the interaction between thisprotein with the viral protein NS2B from Flavivirus (NS4A inHepacivirus), which is necessary for activation of the protease.However, the presence of this segment does not guarantee that peptidesare capable to block the viral infection in vitro and in vivo. Thus, weshow in the example 3 that the segment Ser70-Gly82 corresponding theprotein NS2B from DV2 is capable of inhibiting the viral infection invitro only if it is present in the same polypeptide chain together witha cell penetrating peptide. In order to inhibit the viral infection, thepeptides of the present invention need to penetrate cells, get accessinto the cytosol and bind the NS3pro protein, whose folding take placeat the cytosolic face of the ER membrane.

In an embodiment of the present invention, we use as cell penetratingsegment the sequence corresponding to the FG beta hairpin of domain IIIfrom the envelope protein of DV1-4. In this invention we show that [P]segments based in these sequences are capable carry into the celldifferent peptide cargoes. Previously, it has been shown that cyclicpeptides based on the sequence of the FG hairpin from DV1-2, interactwith the cell receptor LRP1 (aplicación de patente: Métodos y moléculaspara la prevención y el tratamiento de la infección con Flavivirus. CU2006-0091. Huerta V, Chinea G, Fleitas N, Martín A M, Sarria M, GuirolaO, Toledo PG, Sánchez A, Besada V A, Reyes O, Garay H E, Cabrales A,Musacchio A, Padrón G R, González L J).

It is known that the LRP1 receptor interacts and internalize into thecells about 30 natural ligands, among them the pertussis exotoxin A(Herz J, Strickland D K. (2001) LRP: a multifunctional scavenger andsignaling receptor. J Clin Invest. 108:779-84. Kounnas M Z, Morris R E,Thompson M R, FitzGerald D J, Strickland D K, Saelinger C B, 1992. Thealpha 2-macroglobulin receptor/low density lipoprotein receptor-relatedprotein binds and internalizes Pseudomonas exotoxin A. J Biol Chem267:12420-12423). This receptor is expressed in the majority of celltypes, tissues and organs. The DV has also the capacity to infect manycell lines and organs, therefore the use of peptides containing a cellpenetrating peptide based on the sequence of the FG hairpin, is veryfavorable in order to achieve an affective internalization into theinfection susceptible cells. LRP1 expression is high in the liver andthe brain, which are main target organs of diseases caused byFlaviviridae. For example, the viruses from the TBE and JEV complexescause encephalitis and the YFV is mainly viscerotropic and causeshepatitis. For the same reason, this segment would be also effectiveagainst HCV, as present in the anti-HCV peptides described in thepresent invention.

In the particular case of chimerical peptides described in the presentinvention as inhibitors of dengue infection, those modules based on theFG hairpin possess a bifunctional character. Besides the alreadydescribed role as cell penetrating segment, this segment displays alsoanti-DV antiviral activity per se. Previously it has been shown thatpeptides based on the FG hairpin inhibit infection productive cell entryof DV by a mechanism which involves an step occurring after virusadhesion to the plasma membrane (aplicación de patente: Métodos ymoléculas para la prevención y el tratamiento de la infección conFlavivirus. CU 2006-0091. Huerta V, Chinea G, Fleitas N, Martín A M,Sarria M, Guirola O, Toledo P G, Sánchez A, Besada V A, Reyes O, Garay HE, Cabrales A, Musacchio A, Padrón G R, González L J). These peptidesare highly efficient inhibiting viral infection when they are present insolution at the moment of virus entry into the cell. Furthermore, asshown in the example 2, the peptides of the present invention, which donot possess a cell penetrating segment based on the FG hairpin and whoseantiviral effect is based only in their NS3 protease inhibitory modules,are less efficient if they are administered to the media at the sametime as the virus. These peptides (lacking FG hairpin segment) showtheir maximum antiviral activity if they are preincubated with the cellsbefore the virus is added, which is consistent with a mechanism ofinhibition requiring cell penetration and an effective intracellularlocalization in order to inhibit the NS3 protease activation.

Therefore, a novel element of the present invention consists in thecombination of a cell entry inhibitor (which is also a cell penetratingpeptide) and a segment inhibiting the viral protease activation.

Hence, these chimerical peptides possess a biological activity profilewhich is more favorable compared with those peptides based in only oneof these segments, considering the relationship between the moments ofpeptide addition with respect to the beginning of the viral infection.

Cell Penetration and Intracellular Fate. N-Terminal Lipidation andRetention in the ER.

The present invention applies also for the lipidation of the previouslydescribed chimerical peptides. The herein mentioned lipidation typicallyconsists on the myristoylation or the palmitoylation at the N-terminalends of peptides. In this patent as myristoylation we mean the chemicalmodification of the peptides by covalent attachment of the myristic acidCH₃(CH₂)₁₂CO₂H to the N-terminal group of the peptides by means of anamide bond, resulting in the chemical structure CH₃(CH₂)₁₂CO₂—NH—P,where P is the amino acid sequence of the myristoylated peptide.Similarly, the palmitoylation results in the addition of theCH₃(CH₂)₁₄CO₂H palmitic group. As lipidation we also mean herein thecovalent attachment of the lipid chain to the side chains of the aminoacid residues SER and/or TYR, added as N-terminal extensions to thepeptides. In order to exert their antiviral activity, which is basedprimarily on the inhibition of the NS3 protease activation, the peptidesof the present invention need to cross various biological barriers,consisting of diverse membrane systems of the cell.

These peptides need to transit from the extracellular space to theirfinal fate optimal for the antiviral effect, the cytosolic face of theER. In general, lipidation increases peptide lipophilicity, which is afavorable property regarding the interaction with biological membranes.In this invention we have originally combined peptide lipidation withthe addition of some traffic and cellular localization signals(sequences) which enhance the biological activity of peptides. Thus,this design is aimed to increase the efficiency of various stepsinvolved in the manifestation of peptide antiviral activity on thecells: adsorption on the plasma membrane, cell penetration,intracellular traffic/retrograde transport, intracellular localizationon the ER membrane and the interaction with NS3pro protein.

The choice of the chemical nature of the lipid(s) adequate for peptidelipidation is not trivial. One premise of the chimerical peptide designof the present invention, consists in selecting specific lipid(s) forits chemical conjugation to the peptides, in such a way that thischemical modification affect favorably the physicochemical andfunctional properties of the peptides concerning the different processesinvolved in their antiviral action: binding to the plasma membrane, cellpenetration/endocytosis, intracellular traffic/retrograde transport,transport to cytosol and binding to NS3pro. During this process, thepeptides should interact with membranes of different biophysicalproperties, and participate in the transport between differentintracellular compartments. An optimal lipid regarding one individualstep could be detrimental respect to other steps and therefore being notindicated for lipidation of antiviral peptides of the present invention.As an example, considering the interaction with the ER membrane, themonosaturated glycerolipids are potentially favorable. These lipids arecommon in this membrane (Keenan T. W. AND Morrea, D. J. Phospholipidclass and fatty acid composition of Golgi apparatus isolated from ratliver and comparison with other cell fractions. Biochemistry 9: 19-25,1970), which is characterized by its higher fluidity and smallerthickness compared to the plasma membrane rich in sphingolipids, sterolsand disaturated phospholipids. Thus, an unsaturated lipid withrelatively short chain which would be adequate for insertion in the ERmembrane would not be favorable in the plasma membrane. This kind oflipids would localize preferably in the most fluid domains of the plasmamembrane, segregated from domains rich in sphingolipids and cholesterolsuch as the lipid rafts, which are involved in endocytosis. Variousprevious analysis of the endocytic routing of lipid analogs differing inthe nature of their hydrophobic tails have shown that short tailedunsaturated lipids after being endocytosed are efficiently recycled backto the plasma membrane via the endocytic recycling compartment (ERC) andsaturated long tailed lipids are routed through the endocytic way tolate endosomes and lysosomes (Mukherjee, S., Soe, T. T and Maxfield, F.R. 1999. J. Cell Biol., 144, 1271-1284; Koval, M., and R. E. Pagano,1989. J. Cell Biol. 108:2169-2181; Mayor, S., J. F. Presley, and F. R.Maxfield, 1993. J. Cell Biol. 121:1257-1269; Sandhoff, K., and A.Klein., 1994. FEBS Lett. 346:103-107).

Previous studies have reported examples of peptide myristoylationfacilitating cell penetration and the biological activity of peptides attheir corresponding intracellular targets (P. J. Bergman, K. R. Gravitt,C. A. O'Brian, An N-myristoylated protein kinase C-alpha pseudosubstratepeptide that functions as a multidrug resistance reversal agent in humanbreast cancer cells is not a P-glycoprotein substrate, Cancer Chemother.Pharmacol. 40 (1997) 453-456. B. R. Kelemen, K. Hsiao, S. A. Goueli,Selective in vivo inhibition of mitogen-activated protein kinaseactivation using cell-permeable peptides, J. Biol. Chem. 277 (2002)8741-8748. T. Eichholtz, D. B. de Bont, J. de Widt, R. M. Liskamp, H. L.Ploegh, A myristoylated pseudosubstrate peptide, a novel protein kinaseC inhibitor, J. Biol. Chem. 268 (1993) 1982-1986).

However, myristoylation per se does not guarantee peptide penetrationinto the cells. In fact, there are examples of myristoylated peptideswhich do not penetrate into the cells and it has been postulated thatpenetration depends also on the nature of the peptide, being favorableproperties having a positive net charge and a homogeneous distributionof basic residues between acid and hydrophobic residues (Carrigan, C.N., Imperiali, B. 2005, Anal. Biochem. 341 290-298).

The first steps in the interaction of peptides of the present inventionwith cells are the adhesion to the plasma membrane and/or the binding tomolecules present on the membrane. The addition of myristoyl orpalmitoyl groups increases the lipophilicity of peptides of thisinvention, facilitating the peptide binding to the plasma membrane.Besides the presence of the lipid, the peptides of this inventioncontain sequences of cell penetrating peptides, which interact withmolecules present in the membrane. An example is the case of cationiccell penetrating peptides which interact with glycosaminoglycans, inparticular with heparin-like heparan sulfates.

It has been shown that binding to heparan sulfates is essential for cellpenetration by cationic peptides. They can also interact with othernegatively charged molecules of the plasma membrane like anionic lipidsand proteins. Similarly, other peptides which interact with endocyticcell receptors can function as carrier or cell penetrating peptidesfacilitating the entry of cargo molecules into the cells. Lipidation ofpeptides of the present invention therefore increases the bindingaffinity to the plasma membrane providing an additional anchoring site.

In general, myristoylated proteins containing a cluster of basicresidues juxtaposed to a myristoylation signal interact favorably withmembranes rich in cholesterol and sphingolipids (McCabe, J. B., andBerthiaume, L. G. (1999). Functional roles for fatty acylatedamino-terminal domains in subcellular localization. Mol. Biol. Cell 10,3771-3786. McCabe, J. B., and Berthiaume, L. G. (2001). N-TerminalProtein Acylation Confers Localization to Cholesterol,Sphingolipid-enriched Membranes But Not to Lipid Rafts/Caveolae. Mol.Biol. Cell 12, 3601-3617), which are components of lipid rafts involvedin endocytic processes and in the routing of proteins to differentorganelles and specific membranes of the cells (JOOST C. M. HOLTHUIS,THOMAS POMORSKI, RENEÂ' J. RAGGERS, HEIN SPRONG, AND GERRIT VAN MEER.2001. The Organizing Potential of Sphingolipids in IntracellularMembrane Transport. Physiol. Rev. 81, 1689-1723. Simons, K. and Ikonen,E. 1997. Functional rafts in cell membranes. Nature, 387: 569-572).Therefore, the myristoylation is favorable for the capacity of peptidesof the present invention to penetrate cells, in particular thosepeptides containing cationic cell penetrating peptides and/orpolyarginines as signals for retention at the cytosolic face the ERmembrane. In fact, it has been shown that lipidated peptides, containingcertain positive charge can penetrate into the cells (Carrigan, C. N.,Imperiali, B. 2005, Anal. Biochem. 341 290-298).

Detergent resistant specialized microdomains of the membrane (DRMs) richin glycosphingolipids and cholesterol seem to be essential forinternalization of various bacterial toxins into the cells (Choleratoxin, Ricin, Shiga toxin, etc) and molecules associated to these DRMslike the GM1 ganglioside and the sphingolipid Gb3 are receptors of someof these toxins which penetrate cells by endocytosis (Spangler, B. D.(1992) Microbiol. Rev. 56, 622-647. Fujinaga Y, Wolf A A, Rodighiero C,Wheeler T E, Tsai B, Allen L, Jobling M G, Rapoport T A, Holmes R K,Lencer W I. 2003. Gangliosides that associate with lipid rafts mediatetransport of cholera and related toxins from the plasma membrane toendoplasmic reticulum. Mol Biol Cell 14: 4783-4793. Falguieres T,Mallard F, Baron C, Hanau D, Lingwood C, Goud B, Salamero J, Johannes L.2001. Targeting of Shiga toxin B-subunit to retrograde transport routein association with detergent-resistant membranes. Mol Biol Cell 12:2453-2468).

These toxins exert their activities in the cytosol after passing througha retrograde transport process which involves the traffic from theendosomes to the ER, well directly or through the TGN (Sandvig K, vanDeurs B. 2002. Membrane traffic exploited by protein toxins. Annu RevCell Dev Biol 18: 1-24). Then these toxins pass from the ER to thecytosol also by retrograde transport and apparently making use of the ERassociated degradation mechanism (ERAD) (Lord, J. M., and Roberts, L. M.(1998) J. Cell Biol. 140, 733-736. Lord, J. M., Deeks, E., Marsden, C.J., Moore, K., Pateman, C., Smith, D. C., Spooner, R. A., Watson, P.,and Roberts, L. M. (2003) Biochem. Soc. Trans. 31, 1260-1262. AbuJarour,R. J., Dalal, S., Hanson, P. I. and Draper, R. K. 2005. J. Biol. Chem.280, 15865-15871).

Thus, the potential colocalization of the lipidated peptides of thepresent invention in membrane domains rich in sphingolipids would beconsistent with their potential capacity to exploit a cell penetrationmechanism based in the above mentioned lipid dependent retrogradetransport used by bacterial toxins.

In other embodiment of the present invention the designed peptides useas cell penetrating peptide the FG hairpin from domain III of theenvelope protein of DV3 or the homologous peptides of serotypes 1, 2 and4. Previously, it has been shown that these peptides bind to thecellular receptor LRP1 (aplicación de patente: Métodos y moléculas parala prevención y el tratamiento de la infección con Flavivirus. CU2006-0091. Huerta V, Chinea G, Fleitas N, Martín A M, Sarria M, GuirolaO, Toledo P G, Sanchez A, Besada V A, Reyes O, Garay H E, Cabrales A,Musacchio A, Padrón G R, González L J). This receptor mediates theendocytosis of about 30 ligands and it is used for cell entry by thepertussis exotoxin PTx (Herz J, Strickland D K. (2001) LRP: amultifunctional scavenger and signaling receptor. J Clin Invest.108:779-84. Kounnas M Z, Morris R E, Thompson M R, FitzGerald D J,Strickland D K, Saelinger C B. The alpha 2-macroglobulin receptor/lowdensity lipoprotein receptor-related protein binds and internalizesPseudomonas exotoxin A. J Biol Chem 1992; 267:12420-12423).

In this invention we have shown that peptides corresponding to the FGhairpin are capable to mediate the cell entry of peptide cargoes. Thelipidation (myristoylation or palmitoylation) of these FG hairpincontaining peptides would increase the effective affinity for theircellular receptor by enhancing the partition of the peptides in thelipid membrane. This peptide lipidation is also consistent with anincrease in the cell penetrating potential of peptides via endocytosismediated by LRP1 receptor.

One possibility is that these peptides penetrate into the cells in asimilar way to PTx. This toxin get access into the cytosol by retrogradetransport from the endosomes passing successively through the TGN, theER and then to the cytosol. PTx has the capacity to exploit at least tworetrograde transport pathways mediated by LRP1 interaction: a) lipiddependent pathway and b) lipid independent pathway (Smith, D. C.,Spooner, R. A., Watson, P. D., Murray, J. L., Hodge, T. W., Amessou, M.,Johannes, L., Lord, J. M. and Roberts, L. M., 2006. InternalizedPseudomonas Exotoxin A can Exploit Multiple Pathways to Reach theEndoplasmic Reticulum. Traffic, 7: 379-393). The lipid dependent pathwayseems to be related to the localization of a 20% of LRP1 molecules inlipid rafts of the plasma membrane. Those peptides of the presentinvention, which are lipidated and have the capability to interact withLPR1 can potentially exploit more efficiently the lipid dependentpathway, in particular those peptides including a basic cluster ofpolyarginines (added as an ER retention signal) have a favorablecomposition to localize in raft adjacent membrane domains rich incholesterol and sphingolipids.

Other embodiment of the present invention consists in peptides havingthe KDEL signal at the C-terminal end. These peptides are synthesizedwith a carboxylic C-terminal end in order to make functional the KDELsignal for retention at the lumen of the ER (Teasdale, R. D. & Jackson,M. R., 1996. Annu. Rev. Cell Dev. Biol. 12, 27-54). The addition of thissignal to the peptide sequences contributes favorably to theirretrograde transport from the Golgi to ER and the later retention ofthese peptides in the lumen of the ER. Thus, this signal contributes tothe penetration into the cytosol of those peptides which make use atleast partially of the retrograde transport pathway. A higher efficiencyof the transport leads to a higher cytosolic concentration of thepeptides and hence a more potent blocking activity of NS3 proteaseactivation. In the example 3 we have shown that the addition of the KDELsignal to peptides of the present invention can lead to an increase ofthe antiviral activity of the peptides.

The addition of the KDEL signal is valid for peptides of the presentinvention having or not lipids attached in their N-terminal ends. It isconsistent with the fact that this signal is found both in soluble andin type II membrane proteins of the ER. Lipidated peptides of thepresent invention present in the lumen of the ER would adopt a topologysimilar to the type II membrane proteins.

In the case of peptides of the present invention having an FG hairpinrelated cell penetrating segment, the addition of the KDEL signalprovides these peptides with the additional capacity of interfering withthe anterograde transport of the receptor LRP1 and hence leading to adecrease of their receptor expression levels on the plasma membrane.Therefore, the combination of these sequence/signals has an indirectnegative effect on the entry of the virus into the cells reducing theexpression of the receptor at the plasma membrane, and this effect isadditional to the above described direct effect of peptides based on theFG hairpin blocking the cell entry of the virus. Previous evidencesindicate that peptides based on the FG hairpin favor the interaction ofLRP1 with its chaperone receptor associated protein RAP (aplicación depatente: Métodos y moléculas para la prevención y el tratamiento de lainfección con Flavivirus. CU 2006-0091. Huerta V, Chinea G, Fleitas N,Martín A M, Sarria M, Guirola O, Toledo P G, Sánchez A, Besada V A,Reyes O, Garay H E, Cabrales A, Musacchio A, Padrón G R, González L J).It means that these peptides are able to interact with intracellularLRP1, present in the exocytic pathway during its transit to the plasmamembrane. As these peptides contain also the KDEL signal, theintracellular complexes peptide-LRP1 and/or peptide-LRP1/RAP could bindto the KDEL receptor and thus they could be routed from the Golgi to theER, affecting the transport of LRP1 to the plasma membrane andindirectly affecting the LRP1 mediated cell entry of the virus.

A common property of peptides of the present invention is that theydisplay antiviral activity based on inhibition of NS3 proteaseactivation. The inhibition of protease activation is achieved byblocking specifically the interaction of the protein NS2B (NS4A inHepacivirus) with the NS3pro domain, being this interaction a necessarycondition for the correct folding and full activity of the protease.

The protein folding and activation of the protease NS3, as well as thefolding and processing of the core protein and the rest ofnon-structural proteins, takes place at the cytosolic face of the ERmembrane. Therefore, a way to enhance the antiviral activity of thepeptides of the present invention consists in increasing theirintracellular localization at the ER membrane. With this aim, thepeptides could be chemically lipidated (myristoylated or palmitoylated)at the N-terminal end. The lipidated peptides have the capacity tointeract favorably with lipid membranes. The better association of thelipidated peptides with the ER membrane (favored by the lipid moiety)increases the effective apparent affinity of the interaction between thepeptides and the NS3pro protein, an effect related to the followingfactors: 1) increase of the local peptide concentration, 2) thebimolecular interaction occurs in two dimensions (the plane of themembrane) and 3) the fast lateral diffusion of lipidated peptides at themembrane. Furthermore, those peptides lipidated at their N-terminal endwhen associated to the cytosolic face of the ER membrane simulatetopologically the type I membrane proteins, thus they acquires not onlythe correct localization but also an orientation respect to the membranewhich is similar to the viral NS2B protein (NS4A in Hepacivirus).

In general, when there are not additional signals like palmitoylationand/or basic clusters, the myristoylation of cytosolic proteins induce alocalization of these proteins mainly at the ER membrane (McCabe, J. B.,and Berthiaume, L. G. (1999). Functional roles for fatty acylatedamino-terminal domains in subcellular localization. Mol. Biol. Cell 10,3771-3786). The association of the myristate per se with the membrane isnot strong enough and does not guarantee the total retention of peptidesin the ER membrane. However, the ER membrane constitutes the 60% of theintracellular membranes which guarantee a significant effectiveconcentration of the peptides with respect to the rest of membranes.When in addition to myristoylation basic clusters are also present, thecytosolic proteins localize mainly at the inner face of the plasmamembrane and in endosomes.

Various peptides of the present invention contain cationic segments ascell penetrating peptides which guarantee also a favored interactionwith negatively charged molecules located at the outer face of theplasma membrane. Some peptide of the present invention contains clustersof arginines as cationic segment which also constitute ERretention/redirection signals (Teasdale, R. D. & Jackson, M. R. (1996)Annu. Rev. Cell Dev. Biol. 12, 27-54. Zerangue, N., Schwappach, B., Jan,Y. N. & Jan, L. Y. (1999) Neuron 22, 537-548. Schutze, M. P., Peterson,P. A. & Jackson, M. R. (1994) EMBO J. 13, 1696-1705). These peptideshave been designed with the aim to display simultaneously bothproperties: an efficient cell penetration and an intracellularlocalization mainly at the cytosolic face of the ER membrane. Thearginines based traffic signals to the ER are highly efficient and playan important role in the mechanism of quality control of membraneproteins (Chang, X. B., Cui, L., Hou, Y. X., Jensen, T. J., Aleksandrov,A. A., Mengos, A. & Riordan, J. R. (1999) Mol. Cell. 4, 137-142.Margeta-Mitrovic, M., Jan, Y. N. & Jan, L. Y. (2000) Neuron 27, 97-106).

Unlike the dilysine signal which is restricted to the C-terminal end oftype I membrane protein, the arginines based signals are found in manypositions of the sequence of membrane proteins, including the N- andC-terminal ends and also the internal loops located at the cytosolicface. The versatility of the retention signals based in arginines havebeen exploited in the present invention in order to design peptideswhich combine them with the C-terminal KDEL signal. Thus, some lipidatedpeptides of the present invention containing these signals, enter intothe cells and transit to the cytosol by retrograde transport, beingfavored by the KDEL signal during their transit to the ER and laterretained at the cytosolic face of the ER membrane supported by thearginines based signal.

In one embodiment of the present invention we have included the designof lipidated peptides whose sequence contain two successive putativeretention signal for retention at the cytosolic face of the ER membrane.The resulting sequence is LRRRRLRRRRL, which corresponds to twoconsecutive LRRRRL sequence overlapped in a central Leu residue. Thesequence of four consecutive arginine preceded by a hydrophobic residueis typical of RE retention sequences (Zerangue, N., Malan, M. J., Fried,S. R., Dazin, P. F., Jan, Y. N., Jan, L. Y. and Schwappach, B. 2001.PNAS, 98: 2431-2436). In these regards, one of the novel aspects of thepresent invention is that the resulting sequence have the duality ofbeing an efficient RE retention signal and also a cell penetratingpeptide. The cell penetrating property of the resulting sequence isprovided by the eight arginine residues which is similar to thepolyarginine sequences, very efficient cationic PTDs.

DESCRIPTION OF THE FIGURES

FIG. 1: Design of peptide inhibitors of NS3pro protease activation. A:Multiple sequence alignment of NS2B protein sequences SEQ ID NOS:370-382 from Flavivirus. The herein described activation inhibitorsegments are highlighted with double arrows, the light (dark) gray arrowcorresponds to the segment bound to the N-terminal (C-terminal) betabarrel domain of NS3pro. B: Three dimensional structural model of theNS2B-NS3pro complex of Flavivirus. The segment D₅₀-E₆₂ of NS2B from DV2bound the N-terminal beta barrel domains of NS3pro and segment S₇₀-G₈₂of NS2B from DV2 bound to the C-terminal domain are highlighted. C:NS3pro protease activation inhibitory segments D₅₀-E₆₂ andS₇₀-I₈₆-GGGGRR. The C-terminal extension of the latter peptide binds tothe protease active site, blocking the interaction of the protease withits substrates. D: Model of the complex formed by NS3pro in its inactiveconformation (structure of the protein without NS2B) and acomputationally designed peptide of the present invention.

FIG. 2: Assay of inhibition of infection by dengue 2 virus in Verocells. A: Percentage of reduction in the number of plaques due to thepresence of peptide NS2Bden2+TAT with and without preincubation beforeaddition of the virus to the cells. B: Assay of the antiviral activityof peptides TAT and NS2Bden2+TAT at different concentrations, with (pre)and without preincubation (no pre).

FIG. 3: Effect of incubation time on the antiviral activity of peptideNS2Bden2+TAT. PX1: not related negative control peptide (TAT peptidefusion to a not related sequence); P10: peptide NS2Bden2+TAT (TAT fusionto a peptide from NS2B of DV, peptide No. 1 of table 1). The assayedpreincubation times were 0, 30, 60 and 180 minutes.

FIG. 4: Role of internalization on the antiviral activity of peptideNS2Bden2+TAT. A: After preincubation with peptides, they remain presentin the media at the time of virus addition to the cells. B: The peptidesare retired from the media, through various washing before virusaddition to the cells. pNR+TAT: peptide No. 18 of table 1. The pNR+TATpeptide is a negative control of the experiment. Its primary structureis analog to peptide NS2Bden2+TAT, the [I] segment have an amino acidcomposition identical to peptide NS2Bden2+TAT but the sequence waspermutated (peptide 18 of table 1).

FIG. 5: Effect of penetrating peptide identity and ER retention signalon the antiviral activity of peptides. NS2Bden2+TAT: peptide 1 of table1; NS2Bden2+pP2: peptide 2 of table 1, cell penetrating segment ispenetratin; NS2Bden2+pRR: peptide 3 of table 1, decaarginine as cellpenetrating peptide; NS2Bden2+TAT+KDEL: peptide 4 of table 1; pNR+TAT:peptide 18 of table 1, negative control; NS2Bden2: segment [I] ofpeptide NS2Bden2+TAT.

FIG. 6: Antiviral activity of peptides against homologous andheterologous serotypes of DV. The antiviral activity of peptides wastested by reduction of the number of viral plaques in presence of VD1(A), VD3 (B) and VD2 (C). Rosseta: peptide computationally designed tobind to the N-terminal domain of NS3pro of DV2 (peptide 5 of table 1);NS2Bden2+poliR: peptide 3 of table 1, decaarginine as cell penetratingpeptide; NS2Bden2+TAT: peptide 1 of table 1; NS2Bden1+TAT: peptide 6 oftable 1; NS2 Bpermutado+TAT: peptide 18 of table 1, negative control ofthe experiment. The primary structure of NS2 Bpermutado+TAT is analogousto peptide NS2Bden2+TAT, the [I] segment have an amino acid compositionidentical to NS2Bden2+TAT but the sequence was permutated.

EXAMPLES Example 1 Design and Synthesis of Chimerical Peptides Inhibitorof the Infection by Flaviviridae

The chimerical peptides inhibitor of the infection by Flaviviridaedescribed in this invention have a primary structure according to thefollowing topologies:[P]-[L₁]-[I]-[L₂]-[T] o [I]-[L₃]-[P]-[L₄]-[T],where, [P] is the amino acid sequence of a cell penetrating peptide,typically of 10-30 residues, which have the capacity to facilitate theinternalization of the whole peptide molecule into the cell cytoplasmand to get access to the contiguity of the RER; [L1, L2, L3, L4], arelinker sequences of 0-6 residues; [I], is an amino acid sequence whichblocks the activation of NS3pro protease, residues of this segment makecontacts with at least one amino acid from the beta strands B2a and B2bof the C-terminal beta barrel domain, or the beta strand A1 of theN-terminal beta barrel domain of NS3pro protein from Flavivirus (or thestructurally corresponding region in Hepacivirus or Pestivirus), beingthe NS3pro protein in its active or inactive conformation (FIG. 1); [T],sequence of 0 to 10 residues, typically is one or two signals ofretention in the ER like the sequences KDEL and LRRRRL, or the sequenceXRR which displays a capability to binding to the protease active site.

Tables 1 and 2 show sequences of chimerical peptides according to thetopologies 1 and 2 respectively. The basic peptide design is based inthe presence of a protease activation inhibitor segment [I] and a cellpenetrating segment [P]. As [I] segments are included the sequencesD₅₀-E₆₂, S₇₀-G₈₂ and S₇₀-I₈₆ of the NS2B protein from DV1-4. Thecorresponding sequences from WNV and HCV are also included. The segmentD₅₀-E₆₂ binds to the N-terminal domain of NS3pro and the segmentsS₇₀-G₈₂ y S₇₀-I₈₆ bind to the C-terminal domain (FIG. 1A-C).

TABLE 1Design of chimerical peptides according to the topology [P]-[L1]-[I]-[L2]-[T]SEQ   ID Penetrating Target NO. No [P] [L1] [I] [L2] [T] Virus peptidedomain 1 1 YGRKKRRQRRRPPQ GGG SSPILSITISEDG dengue 2 TAT C- terminal 5 2RQIKIWFQNRRMKWKK GGG SSPILSITISEDG dengue 2 penetratin  C- terminal 6 3RRRRRRRRRR GGG SSPILSITISEDG dengue 2 R10 C- terminal 16 4YGRKKRRQRRRPPQ GGG SSPILSITISEDG GG KDEL* dengue 2 TAT C- terminal 26 5YGRKKRRQRRRPPQ GGG QWPALPKIEAQDG diserio TAT N- terminal 27 6YGRKKRRQRRRPPQ GGG ASRNILVEVQDDG denguel TAT C- terminal 28 7YGRKKRRQRRRPPQ GGG VSRNLMITVDDDG dengue3 TAT C- terminal 29 8YGRKKRRQRRRPPQ GGG SSPIIEVKQDEDG dengue4 TAT C- terminal 20 9YGRKKRRQRRRPPQ GGG SSERVDVRLDDDG WNV TAT C- terminal 31 10YGRKKRRQRRRPPQ bA SSPILSITISEDG GGG GRR* dengue 2 TAT C- SMSI terminal39 11 YGRKKRRQRRRPPQ GGG DLELERAADVKWE dengue 2 TAT N- terminal 47 12RRRRRRRRRR GGG DLELERAADVKWE dengue 2 R10 N- terminal 55 13YGRKKRRQRRRPPQ GGG DLELERAADVKWE GG KDEL* dengue 2 TAT N- terminal 56 14YGRKKRRQRRRPPQ GGG DLSLEKAAEVSWE denguel TAT N- terminal 57 15YGRKKRRQRRRPPQ GGG DLTVEKAADVTWE dengue3 TAT N- terminal 58 16YGRKKRRQRRRPPQ GGG DL SLEKAANVQWD dengue4 TAT N- terminal 59 17YGRKKRRQRRRPPQ GGG DMWIERTADITWE WNV TAT N- terminal 63 18YGRKKRRQRRRPPQ GGG LEGSDISPSTISI negative   TAT control 64 19YGRKKRRQRRRPPQ negative   TAT control 65 20 YGRKKRRQRRRPPQ GGGTGSVVIVGRIIL HCV TAT N- terminal 66 21 YGRKKRRQRRRPPQ GGG TGSVVIVGQIILHCV TAT N- terminal 67 22 CSNIVIGIGDKALKINWC bA SSPILSITISEDG dengue 2FG-den3 C- terminal 77 23 CSNIVIGIGDKALKINWC bA DLELERAADVKWE dengue 2FG-den3 N- terminal 85 24 CSNIVIGIGDKALKINWC bA SSPILSITISEDG   GGG GRR*dengue 2 FG-den3 C- SMSI terminal 93 25 CSNIVIGIGDKALKINWC bASSPILSITISEDG GG KDEL* dengue 2 FG-den3 C- terminal 103 26CSNIVIGIGDKALKINWC bA DLELERAADVKWE GG KDEL* dengue 2 FG-den3 N-terminal 111 27  myr-bA-CSNIVIGIGDKALKINWC bA SSPILSITISEDG bA LRRRRLdengue 2 FG-den3 C- terminal 121 28  myr-bA-CSNIVIGIGDKALKINWC bADLELERAADVKWE bA LRRRRL dengue 2 FG-den3 N- terminal 111 29pal-bA-CSNIVIGIGDKALKINWC bA SSPILSITISEDG bA LRRRRL dengue 2 FG-den3 C-terminal 121 30 pal-bA-CSNIVIGIGDKALKINWC bA DLELERAADVKWE bA LRRRRLdengue 2 FG-den3 N- terminal 129 31  myr-bA-CSNIVIGIGDKALKINWC bASSPILSITISEDG dengue 2 FG-den3 C- terminal 139 32 myr-bA-CSNIVIGIGDKALKINWC bA DLELERAADVKWE dengue 2 FG-den3 N- terminal129 33 pal-bA-CSNIVIGIGDKALKINWC bA SSPILSITISEDG dengue 2 FG-den3 C-terminal 139 34 pal-bA-CSNIVIGIGDKALKINWC bA DLELERAADVKWE dengue 2FG-den3 N- terminal 147 35  myr-bA-CSNIVIGIGDKALKINWC bA SSPILSITISEDGbA LRRRRLKDEL* dengue 2 FG-den3 C- terminal 148 36 myr-bA-CSNIVIGIGDKALKINWC bA DLELERAADVKWE bA LRRRRLKDEL* dengue 2FG-den3 N- terminal 147 37 pal-bA-CSNIVIGIGDKALKINWC bA SSPILSITISEDG bALRRRRLKDEL* dengue 2 FG-den3 C- terminal 148 38pal-bA-CSNIVIGIGDKALKINWC bA DLELERAADVKWE bA LRRRRLKDEL* dengue 2FG-den3 N- terminal 364 39 RRRRRRRRRR GGG SSPILSITISEDG GG KDEL*dengue 2 R10 C- terminal *: carboxilic C-terminal end. Myr-: covalentattachment of a myristoyl group to the N-terminal end of the peptide.Pal-: covalent attachment of a palmitoyl group to the N-terminal end ofthe peptide. bA: beta-Alanine. FG-den3: sequence corresponding to the FGhairpin of domain III of the envelope protein from DV3, two cysteinsbound by disulfide bridge are added at the N- and C-terminal ends of thesegment.

TABLE 2 Design of chimerical peptides according to the topology [I]-[L3]-[P]-[L4]-[T]SEQ ID    Penetrating Target NO. No [I] [L3] [P] [L4] Virus Peptidedomain 158 1 SSPILSITISEDG GGG YGRKKRRQRRRPPQ dengue2 TAT C-terminal 1592 SSPILSITISEDG GGG RRRRRRRRRR dengue2 R10 C-terminal 167 3SSPILSITISEDG GGG RRRRRRRRRR GG KDEL* dengue2 R10 C-terminal 175 4SSPILSITISEDG GGG YGRKKRRQRRRPPQ GG KDEL* dengue2 TAT C-terminal 183 5ASHNILVEVQDDG GGG YGRKKRRQRRRPPQ dengue1 TAT C-terminal 184 6VSHNLMITVDDDG GGG YGRKKRRQRRRPPQ dengue3 TAT C-terminal 185 7SSPIIEVKQDEDG GGG YGRKKRRQRRRPPQ dengue4 TAT C-terminal 182 8SSERVDVRLDDDG GGG YGRKKRRQRRRPPQ WNV TAT C-terminal 190 9 DLELERAADVKWEGGG YGRKKRRQRRRPPQ dengue2 TAT N-terminal 200 10 DLELERAADVKWE GGGYGRKKRRQRRRPPQ GG KDEL* dengue2 TAT N-terminal 210 11 DLELERAADVKWE GGGRRRRRRRRRR dengue2 R10 N-terminal 211 12 DLELERAADVKWE GGG RRRRRRRRRR GGKDEL* dengue2 R10 N-terminal 221 13 DLSLEKAAEVSWE GGG RRRRRRRRRR Dengue1R10 N-terminal 222 14 DLTVEKAADVTWE GGG RRRRRRRRRR Dengue3 R10N-terminal 223 15 DLSLEKAANVQWD GGG RRRRRRRRRR Dengue4 R10 N-terminal224 16 DMWIERTADITWE GGG RRRRRRRRRR WNV R10 N-terminal 230 17SSPILSITISEDG bA LRRRRLbALRRRRL bA KDEL* dengue2 2(LR4L) C-terminal 23818 SSPILSITISEDG bA LRRRRLbALRRRRL dengue2 2(LR4L) C-terminal 246 19SSPILSITISEDG bA LRRRRLRRRRL dengue2 2(LR4L) C-terminal 254 20 myr- bALRRRRLRRRRL dengue2 2(LR4L) C-terminal SSPILSITISEDG 254 21 pal- bALRRRRLRRRRL dengue2 2(LR4L) C-terminal SSPILSITISEDG 262 22 myr- bALRRRRLbALRRRRL bA KDEL* dengue2 2(LR4L) C-terminal SSPILSITISEDG 270 23myr- bA LRRRRLbALRRRRL dengue2 2(LR4L) C-terminal SSPILSITISEDG 262 24pal- bA LRRRRLbALRRRRL bA KDEL* dengue2 2(LR4L) C-terminal SSPILSITISEDG270 25 pal- bA LRRRRLbALRRRRL dengue2 2(LR4L) C-terminal SSPILSITISEDG278 26 DLELERAADVKWE bA LRRRRLbALRRRRL bA KDEL* dengue2 2(LR4L)N-terminal 288 27 myr- bA LRRRRLbALRRRRL bA KDEL* dengue2 2(LR4L)N-terminal DLELERAADVKWE 288 28 pal- bA LRRRRLbALRRRRL bA KDEL* dengue22(LR4L) N-terminal DLELERAADVKWE 298 29 DLELERAADVKWE bA LRRRRLRRRRLdengue2 2(LR4L) N-terminal 299 30 myr- bA LRRRRLRRRRL dengue2 2(LR4L)N-terminal DLELERAADVKWE 299 31 pal- bA LRRRRLRRRRL dengue2 2(LR4L)N-terminal DLELERAADVKWE 309 32 DLSLEKAAEVSWE bA LRRRRLRRRRL Dengue12(LR4L) N-terminal 310 33 DLTVEKAADVTWE bA LRRRRLRRRRL Dengue3 2(LR4L)N-terminal 311 34 DLSLEKAANVQWD bA LRRRRLRRRRL Dengue4 2(LR4L)N-terminal 318 35 DLELERAADVKWE bA CSNIVIGIGDKALKINWC dengue2 FG-den3N-terminal 328 36 myr- bA CSNIVIGIGDKALKINWC bA LRRRRL dengue2 FG-den3N-terminal DLELERAADVKWE 328 37 pal- bA CSNIVIGIGDKALKINWC bA LRRRRLdengue2 FG-den3 N-terminal DLELERAADVKWE 338 38 myr- bACSNIVIGIGDKALKINWC bA LRRRRLKDEL* dengue2 FG-den3 N-terminalDLELERAADVKWE 338 39 pal- bA CSNIVIGIGDKALKINWC bA LRRRRLKDEL* dengue2FG-den3 N-terminal DLELERAADVKWE 339 40 SSPILSITISEDG bACSNIVIGIGDKALKINWC dengue2 FG-den3 C-terminal 347 41 myr- bACSNIVIGIGDKALKINWC bA LRRRRL dengue2 FG-den3 C-terminal SSPILSITISEDG347 42 pal- bA CSNIVIGIGDKALKINWC bA LRRRRL dengue2 FG-den3 C-terminalSSPILSITISEDG 355 43 myr- bA CSNIVIGIGDKALKINWC bA LRRRRLKDEL* dengue2FG-den3 C-terminal SSPILSITISEDG 355 44 pal- bA CSNIVIGIGDKALKINWC bALRRRRLKDEL* dengue2 FG-den3 C-terminal SSPILSITISEDG *: carboxilicC-terminal end. Myr-: covalent attachment of a myristoyl group to theN-terminal end of the peptide. Pal-: covalent attachment of a palmitoylgroup to the N-terminal end of the peptide. bA: beta-Alanine. FG-den3:sequence corresponding to the FG hairpin of domain III of the envelopeprotein from DV3, two cysteins bound by disulfide bridge are added atthe N- and C-terminal ends of the segment.

The present invention concerns also the design of antiviral chimericalpeptides against the other members of the Flaviviridae family. Peptideinhibitors against other Flaviviridae include as [I] segments, theanalogous segments from the corresponding NS2B protein sequence (inFlavivirus) or NS4A (in hapacivirus). In the list of sequences of thepresent invention we include additional chimerical peptides analogous tothose shown on tables 1 and 2, whose [I] segment corresponds to otherFlavivirus (YFV, JEV, TBE, WNV) and the Hepacivirus HCV.

As [P] segments we consider the TAT peptide, R10, penetratin, thecationic sequences LRRRRLRRRRL (SEQ ID NO: 366) and LRRRRL-bAla-RRRRL(SEQ ID NO: 365) and the segment 5376-W391 of the envelope protein ofDV3 (loop FG of domain III). The later segment includes cysteines at itsN- and C-terminal ends, which form a disulfide bridge and stabilize thebeta hairpin conformation observed in the three dimensional structure ofthe envelope protein.

As terminal [T] segments we include the ER retention signals LRRRRL (SEQID NO: 367), KDEL and their combination LRRRRLKDEL (SEQ ID NO: 368). Thepresence of these signals enhances the effective localization ofpeptides in the ER, which affect favorably their antiviral activity. Wealso include as [T] segment the sequence GRR, linked by the tripeptideGGG to the [I] segment of sequence S₇₀-I₈₆. As shown in FIG. 1C,peptides with this primary structure bind to the C-terminal domain ofNS3pro protein and the GRR segment localizes at the protease activesite, blocking its interaction with substrates. As linker segments weinclude in table 1 and 2 the tripeptide GGG, the dipeptide GG and theamino acid beta-Alanine.

Peptides myristoylated and palmitoylated at the N-terminal end are alsoincluded. The lipidation of these peptides increases the efficiency ofthe adhesion to the plasma membrane, cell entry and the finallocalization in the RE membrane. Lipidation is carried out by chemicalmethods. In the table 1 and 2, the lipids are attached directly to theN-terminal ends or to an N-terminal beta-Alanine residue.

Various peptides segments included in table 1 and 2 display more thanone single function. The segments LRRRRLRRRRL (SEQ ID NO: 365) andLRRRRL-bAla-RRRRL (SEQ ID NO: 366) besides being cell penetratingpeptides comprise two consecutive ER retention signals.

The [P] segment corresponding to the sequence of the region S376-W391 ofthe envelope protein from DV3, besides being a cell penetrating peptide,is also an inhibitor of the virus entry into the cells. Therefore theuse of this segment in peptides of the present invention increases theinhibitory effect of these peptides.

The peptides of the present invention could be obtained by chemicalsynthesis o by recombinant DNA technology, alone or as part of fusionproteins. Expression as fusion proteins can increase the expressionlevels and stability of peptides against degradation by host proteases.These peptide sequences could be joined to the fusion proteins throughlinkers corresponding to substrate sequences of specific proteases, andthus the peptides can be isolated by successive proteolysis andpurification.

Peptide Synthesis

Solid phase peptide synthesis was performed on an Fmoc-AM-MBHA resin,using the Fmoc/tBu strategy (Barany, G. and Merrifield, R. B. J Am Chem.Soc. 99 (1977) 7363-7365). The synthesis was carried out manually in 10ml syringes equipped with porous frit and all reactive and solvents werediscarded by vacuum filtration. The amino acids were coupled byactivation with DIC/HOBt, monitoring the completion of the couplingreaction by the ninhydrin assay (Kaiser, E., Colescott, R. L.,Bossinger, C. D., Cook, P. I. Anal Biochem. 34 (1970) 595-598).

The synthesized peptides were detached from the resin by treatment witha solution of TFA/EDT/H2O/TIS (94%/2.5%/2.5%/1%), precipitated withether, and lyophilized during 72 h. Peptide cyclization by forming adisulphide bridge was achieved by oxidation with DMSO (Andreu, D.,Albericio, F., Sol, N. A., Munson, M. C., Ferrer, M. and Barany, G.,Pennington, M. W. and Dunn, B. M. (Eds), Peptide Synthesis Protocols,Methods in Molecular Biology, Totowa, N.J., 1994, pp. 91-169). In allcases, the peptides were purified by RP-HPLC and the collected fractionswere analyzed again by analytical RP-HPLC. The final preparation of eachpeptide was obtained by pooling the fractions with a chromatographicpurity equal to or higher than 99%. The mass of the peptide on eachfinal preparation was verified by ESI-MS mass spectrometry.

The mass spectra were acquired with a hybrid mass spectrometer withoctagonal geometry QTOF-2™ (Micromass, UK), equipped with a Z-sprayelectronebulization ionization source.

The software used for the acquisition and processing of the spectra wasMassLinx, ver. 3.5 (Waters, USA).

Example 2 Inhibition of Viral Infection in Vero Cells

In order to prove the antiviral activity in vitro of chimerical peptidesdescribed on the present invention, the peptides were tested in plaquereduction neutralization assay in Vero cells (PRNT). Vero cells weregrown in 24-well plates to approximately 90% confluence, and washedtwice with MEM medium without FCS. Peptide dilutions were addedaccording to the particular assay and incubated typically during 1 h at37° C. After the incubation, the virus was added at a multiplicity ofinfection of 0.1, followed by a subsequent incubation for 1 hour at 37C. In certain experiments the peptides were added simultaneously withthe virus (without preincubation) or the peptide preincubation time wasmodified. At the end of the second incubation, the unbound virus waseliminated by washing, and the cells were incubated for 5 days at 37 Cin high density medium (MEM supplemented with non essential amino acids,1% FCS, 1% carboxymethylcellulose) in order to propitiate the appearanceof lytic plaques. The plaques were visualized by staining with 0.1%Naphtol Blue Black in 0.15 Mol/L sodium acetate. Two replicates wereused per experimental point in each assay, and three independentdeterminations were performed. The inhibition percentage was calculatedaccording to the expression:

$100{x\left\lbrack {1 - \frac{{No}.{plaques}}{{No}.{Plaques}.{virus}.{control}.}} \right\rbrack}$

The FIG. 2 shows that peptide NS2Bden2+TAT (peptide 1 of the table 1)inhibit the infection by DV2, in a dose dependent manner, with a 1050 ofapproximately 50-60 μM. The peptides showed no signs of toxicity on thecells at the assayed conditions. The sequence of peptide NS2Bden2+TATcontains two essential modules: the cell penetrating segment TAT and theprotease NS3pro activation inhibitor segment, which targets theC-terminal domain of the protease. The TAT peptide did not showantiviral activity and caused an increase in the number of plaques (FIG.2B). This result is consistent with the peptide design: the antiviralactivity residing on the segment specifically related to the VD2 (strainS16803). The observed enhancement of the virus infection in presence ofthe TAT peptide could be related to an increase in the entry of thevirus into the cells facilitated by the PTD property of this peptide.Increasing cell penetration of viruses mediated by PTD has beenpreviously observed in other systems (Gratton J P, Yu J, Griffith J W,et al. Cell-permeable peptides improve cellular uptake and therapeuticgene delivery of replication-deficient viruses in cells and in vivo. NatMed 2003; 9: 357-63).

The presence of the TAT peptide segment in the sequence of peptideNS2Bden2+TAT is necessary for its antiviral activity, because the NS3proprotease activation inhibitor segment [I] per se does not show antiviralactivity in vitro (FIG. 5).

The FIG. 2 shows that the antiviral activity of the chimerical peptideNS2Bden2+TAT increases if the peptide is preincubated with cells 1 hbefore the addition of the virus. This result is consistent with thefact that the target for the antiviral activity of the peptide is anintracellular event, and preincubation allows a higher amount of peptideto penetrate into the cells and localize at the ER membrane, previouslyto the beginning of the virus replication.

In order to characterize the effect of preincubation on the antiviralactivity of peptide NS2Bden2+TAT we studied the relationship betweenplaque reduction neutralization, preincubation time and peptide dose. Asnegative control we used a non related chimerical peptide displaying anstructure similar to NS2Bden2+TAT. This peptide contains at theN-terminal end the sequence of the TAT peptide and at its C-terminal enda sequence which have been shown to bind to the protein E7 from humanpapilloma virus. The FIG. 3 shows that for peptide concentrations lessthan 100 μM, preincubation is necessary for the antiviral activity andthis activity increases with the time of preincubation between 0 and 1hour. This result is consistent with the intracellular localization ofthe target for the antiviral effect and the need for peptide transportfrom the extracellular space to the cytosol.

However, between 1 and 3 hours of preincubation, we do not observe moredifferences. One possible explanation could be that at these timesequilibrium is reached between the kinetics of accumulation of peptidein the cytosol and the intracellular degradation of the peptide.

The negative control peptide does not show antiviral activity at any ofthe assayed conditions indicating that the antiviral effect of thepeptide NS2Bden2+TAT is due specifically to the sequence of the segmentcorresponding to the NS2B protein.

FIG. 4 shows additional evidence indicating that the antiviral activityof the peptide NS2Bden2+TAT is related to an intracellular effect. Inthis case, besides the previously described usual assay conditions (FIG.4A), the antiviral activity of the peptide was also determined when thepeptide was retired from the media by successive washing of cellsprevious to the addition of the virus (FIG. 4B). In both conditions ofthe assay, the antiviral activity of the peptide was very similar,indicating that the antiviral effect depends on previously internalizedpeptide. In these assays, the peptide 18 of table 1 was used as negativecontrol. This peptide has a design similar to the peptide NS2Bden2+TAT,but the C-terminal segment consists of a sequence of the same length andamino acid composition as the NS3pro protease activation inhibitor [I]segment of NS2Bden2+TAT, but the original sequence was randomized. Thispeptide did not show antiviral activity in any condition, indicatingthat the antiviral activity of NS2Bden2+TAT depends on the selectedsequence fragment of NS2B.

Example 3 Effect of the Nature of the Cell Penetrating Peptide and theER Retention Signal on the Antiviral Activity of Peptides

In order to determine the role of the cell penetrating peptide and theER retention signal on the antiviral activity of peptides of the presentinvention we tested peptides No 1, 2, 3 and 4 of table 1 for inhibitionof the viral infection by VD2 in Vero cells, using the assay describedin the example 2.

The peptides 2 and 3 have a primary structure similar to the peptideNS2Bden2+TAT (péptido 1), but displaying penetratin and decargininerespectively as cell penetrating segments. The peptide 4 consists on theaddition of the KDEL signal at the C-terminal end of peptideNS2Bden2+TAT. The C-terminal group of peptide 4 is carboxylic in orderto make functional the ER retention signal.

The FIG. 5 and the table 3 show that the peptide NS2Bden2-pRR (peptide 3on table 1) displays the higher antiviral activity, almost an order morepotent than the peptide NS2Bden2+TAT. One possible explanation is thatdecaarginine peptide is more resistant to proteolysis in theintracellular environment of the cell ((Fischer, R., Kohler, K.,Fotin-Mleczek, M., and Brock, R. 2004. A stepwise dissection of theintracellular fate of cationic cell-penetrating peptides. J. Biol. Chem.279, 12625-35). The peptide NS2Bden2-pP2 (peptide 2) shows an antiviralactivity similar to NS2Bden2+TAT, however it displays significantcytotoxicity. The addition of the KDEL signal increase slightly theantiviral activity of the peptide, suggesting that the peptideNS2Bden2+TAT uses at least partially the retrograde transport to getaccess into the cytosol.

The peptide NS2Bden2 which lacks the cell penetrating segment does notinhibit the antiviral infection, showing that the inclusion of this kindof segment is required in the peptides of the present invention.

TABLE 3 PRNT50 and cytotoxicity (CTE) of peptides in Vero cells PeptidePRNT50 CTE NS2Bden2 + TAT 60 μM 150 μM NS2Bden2 + TAT + KDEL 40 μM >150μM NS2Bden2-pRR <10 μM   >50 μM NS2Bden2-pP2 75 μM 50 μM NS2Bden2 — —CTE: cytotoxic effect, the values indicate peptide concentrationscausing damage to 50% of the monolayer.

Example 4 Antiviral Activity of Peptides Against Homologous andHeterologous Virus

An expected property of antiviral agents is to possess a wide spectrumof antiviral activity, at least against the related most similarviruses. This is also the case in the development of antiviral moleculesagainst dengue virus: 1) dengue is actually a complex of four differentviruses, 2) there are difficulties for an early specific diagnosis and3) in the affected countries, dengue is frequently endemic, occurringthe cocirculation of more that one serotype.

The four dengue serotypes are related viruses with similar amino acidsequences (70-80% identity) of their structural and non structuralproteins. Therefore, it is reasonable that differences in the amino acidsequences of NS2B and/or NS3pro could affect the infection inhibitorycapacity of peptides of the present invention against the heterologousviruses.

In order to evaluate the cross-reactivity or serotype specificity of theantiviral activity of peptides of the present invention, we testedpeptides 1, 3 and 6 of the table 1 for inhibition of the viral infectionby DV1-3 in Vero cells, using the assay described in the example 2. Thetested viral strains were West Pac 74 of DV1, S16803 of DV2 and CH53489of DV3. The peptide 6 (NS2Bden1+TAT) has a primary structure similar toNS2Bden2+TAT, but it has a NS3 activation inhibitor segmentcorresponding the protein NS2B from DV1. We also included in theanalysis the peptide 5, designed by computational methods.

The FIG. 6 shows that the peptide NS2Bden2+pRR (peptide 3 on table 1) isequally potent against the three serotypes. The peptide NS2Bden2+TATalso inhibits the serotypes 1-3 although with a lower antiviralactivity. The peptide NS2Bden1+TAT (peptide 6) however shows onlypartial inhibition against serotypes 1 and 3. This result is consistentwith the fact that serotypes 1 and 3 are phylogenetically closer to eachother and their proteins are more similar.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing.txt”,created on May 3, 2013. The sequence_listing.txt file is 384 kb in size.

The invention claimed is:
 1. A chimerical peptide having a primarystructure[P]-[L₁]-[I]-[L₂]-[T] or [I]-[L₃]-[P]-[P]-[L₄]-[T], wherein [P] is acell penetrating peptide, [L₁]-[L₂]-[L₃] and [L₄] are linker sequencesof 0-6 amino acids, [I] is an NS3pro activation inhibitor sequence whichbinds with at least one amino acid from the beta strands B2a and B2b ofthe C-terminal beta barrel or from the beta strand Al of the N-terminalbeta barrel of the NS3pro protein from a virus of the Flaviviradaefamily, [T] is an amino acid sequence between 0-10 residues capable ofbinding to a P1 and P2 substrate binding sites of the NS3pro proteasefrom a virus of the Flaviviradae family, wherein said chimeric peptideis selected from the group consisting of SEQ ID NOS: 1-62 and 65-364,and wherein said peptide is able to inhibit or attenuate the infectionby the virus.
 2. The peptide according to the claim 1, wherein saidinhibitor sequence is a peptide able to bind to NS3pro and contacts atleast one residue comprised in the region Gly21-Lys28 of the N-terminaldomain of the NS3pro protein from DV2 or structurally equivalentresidues of the NS3pro protein from other Flavivirus.
 3. The chimericalpeptide according to the claim 1, wherein said inhibitor sequence is apeptide able to bind to NS3pro and contacts at least one residuecomprised in the region Gly114-Thr118, Ser127 and Val162 of theC-terminal domain of the NS3pro protein from DV2 or structurallyequivalent residues of the NS3pro protein from other Flavivirus.
 4. Thechimerical peptide according to the claim 1, wherein said inhibitorsequence contacts at least one residue comprised in the regionGlu32-Thr38 of the N-terminal domain of the NS3pro protein from HCV orstructurally equivalent residues of the NS3pro protein from otherHepacivirus.
 5. The chimerical peptide according to the claim 1, whereinsaid inhibitor sequence includes region Asp50-Glu62 of the NS2B proteinfrom DV2 or a structurally equivalent segment of the protein NS2B fromother Flavivirus.
 6. The chimerical peptide according to the claim 1wherein said inhibitor sequence includes NS3pro protease which comprisethe region Ser70-Gly82 of the NS2B protein from DV2 or a structurallyequivalent segment of the protein NS2B from other Flavivirus.
 7. Thechimerical peptide according to the claim 1, wherein said inhibitorsequence includes region Thr20-Leu31 of the NS4A protein from HCV or astructurally equivalent segment of the protein NS4A from otherHepacivirus.
 8. The chimerical peptide according to the claim 1, whereinsaid virus from the Flaviviridae family is one of the followingFlavivirus: West Nile virus, St Louis Encephalitis virus, DV1, DV2, DV3,DV4, Japanese Encephalitis virus, Yellow Fever virus, Kunjin virus,Kyasanur Forest Disease virus, Tick-borne Encephalitis virus, MurrayValley virus, LANGAT virus, Louping ill virus, or Powassan virus.
 9. Thechimerical peptide according to the claim 1, wherein said virus from theFlaviviridae family is a Hepacivirus.
 10. The chimerical peptideaccording to the claim 1, wherein said cell penetrating peptide isselected from one of the following cationic protein transductiondomains: TAT peptide, heptaarginine, octaarginine, nonaarginine,decaarginine, or a peptide identified as SEQ ID NO: 366 or SEQ ID NO:365.
 11. The chimerical peptide according to the claim 1, wherein saidcell penetrating peptide comprises region Ser376-Trp391 of the envelopeprotein from DV2 or a structurally equivalent segment from otherserotype of dengue virus.
 12. The chimerical peptide according to theclaim 1, further comprising a signal of retention and/or localization inthe endoplasmic reticulum wherein said signal increases the antiviralactivity of the peptide.
 13. The chimerical peptide according to theclaim 1, wherein said peptide is lipidated and the lipidation increasesthe antiviral activity of the peptide.
 14. A pharmaceutical compositioncomprising a pharmaceutically acceptable excipient and one or morepeptides according to claim 1, said pharmaceutical composition beingefficacious for treatment against infection by one or more viruses fromthe Flaviviridae family.
 15. The chimerical peptide according to claim9, wherein said Hepacivirus is HCV.